Quinolone chalcone compounds and uses thereof

ABSTRACT

The present disclosure relates to novel compounds, compositions comprising these compounds, and their use, for example for the treatment of cancer. In particular, the present disclosure includes compounds of Formula (I), and compositions and uses thereof.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 62/258,033, filed Nov. 20, 2015, the content ofwhich is hereby incorporated by reference in its entirety.

FIELD

The present disclosure relates to novel quinolone chalcone compounds,compositions comprising such novel quinolone chalcone compounds, andtheir use, for example, for the treatment of cancer.

BACKGROUND

Continuously dividing cancer cells are dependent upon the rapid anddynamic process of the polymerisation and depolymerisation of tubulin.

Microtubule targeting agents such as paclitaxel and vinblastine havebeen widely used at clinics (Dumontet and Jordan, 2010; Kuppens, 2006;Singh et al., 2008). Microtubule targeting agents are known to bindtubulin via at least four different binding sites/areas. Paclitaxelbinds to the inner surface of the β-subunit of polymerized tubulin,resulting in the stabilization of microtubule structure and thuspreventing depolymerisation (Lu et al., 2012). The Laulimalides causemicrotubule stabilization similar to taxanes, although they bind to adifferent site (Pryor et al., 2002). Vinca alkaloids bind to a fewtubulin subunits at the end of the polymer, preventing them fromundergoing polymerisation. However, vinblastine is also capable ofbinding at the interface of two αβ-tubulin heterodimers, thus preventingself-association (Gigant et al., 2005). The fourth group of microtubuletargeting agents bind to tubulin through the colchicine binding site.This class of compounds binds to the β-tubulin subunit, resulting in theinhibition of microtubule assembly (Ravelli et al., 2004). Although itinhibits microtubule assembly, the therapeutic value of colchicine islimited due to its low therapeutic index (i.e., high toxicity).

Unlike taxanes and vinca alkaloids, agents targeting the colchicinebinding site have minimal multidrug resistance issues. Therefore, manyefforts have been undertaken to develop drugs that effectively bind tothe colchicine binding site with minimal side effects (Borisy andTaylor, 1967a; Borisy and Taylor, 1967b; Lu et al., 2012; Weisenberg etal., 1968; Zhou and Giannakakou, 2005). However, to date, an effectivedrug targeting the colchicine-binding site with low side effects has notyet been approved by US FDA.

SUMMARY

It was found that the quinolone chalcones of the studies disclosedherein, as exemplified by the compounds CTR-17[(E)-3-(3-(2-methoxyphenyl)-3-oxoprop-1-enyl) quinolin-2(1H)-one] andCTR-20 [(E)-6-methoxy-3-(3-(2-methoxyphenyl)-3-oxoprop-1-enyl)quinolin-2(1H)-one], bind to tubulin at the colchicine binding site,leading to cell killing in a cancer-specific manner. Both of these CTRcompounds were observed to effectively kill multidrug-resistant(including paclitaxel-, vinblastine- and colchicine-resistant) cancercells. Furthermore, the data obtained in the present studies also showedthat the combination of paclitaxel and CTR-17 or CTR-20 has strongsynergistic effects on multidrug-resistant cells. The data from animalstudies showed that the CTR compounds tested, alone or in combinationwith paclitaxel, possess strong antitumor activity without notableill-effects to animals observed. Further, CTR-21((E)-8-methoxy-3-(3-(2-methoxyphenyl)-3-oxoprop-1-enyl)quinolin-2(1H)-one) and CTR-32((E)-3-(3-(2-ethoxyphenyl)-3-oxoprop-1-enyl)quinolin-2(1H)-one) are alsohighly potent in tumor cell killing. Like CTR-17 and CTR-20, CTR-21 andCTR-32 also disrupt the microtubule dynamics. Data from three isogeniccell lines showed that CTR-20, CTR-21 and CTR-32 preferentially kill thefully malignant MCF10CA1a breast cancer cells over premalignant MCF10AT1and the non-malignant MCF10A breast cells. Furthermore, all of thesecompounds effectively kill multidrug-resistant cancer cells.

Accordingly, the present disclosure includes a compound of Formula I:

wherein

A is O or S;

n is 0, 1, 2 or 3;

when n is 1, R¹ is halo, C₁₋₆alkyl, C₂₋₆alkenyl or —X—C₁₋₆alkyl;

when n is 2 or 3, each R¹ is independently halo, C₁₋₆alkyl, C₂₋₆alkenylor —X—C₁₋₆alkyl; or two R¹ together form a methylenedioxy group that isattached to two adjacent ring carbon atoms;

R² is C₁₋₆alkyl or C₁₋₆haloalkyl;

R³ is absent or is halo, —X—C₁₋₆alkyl or —X—C₁₋₆haloalkyl; and

each X is independently O or S,

or a pharmaceutically acceptable salt, solvate and/or prodrug thereof.

In an embodiment, A is O.

In an embodiment, R³ is absent.

In an embodiment, R² is methyl.

In an embodiment, n is 1 and R¹ is 6-OCH₃, 7-OCH₃, 8-OCH₃, 6-OC₂H₅,6-SCH₃, 7-SCH₃, 6-CH₃, 6-C₂H₅, 6-F, 6-Cl, 6-Br, 7-F, 7-Cl or 7-Br. Inanother embodiment, n is 1 and R¹ is 6-CH₃, 6-OCH₃ or 7-OCH₃.

In an embodiment, n is 2 and R¹ is 6,7-diCH₃, 6,7-diOCH₃ or6,7-O—CH₂—O—. In another embodiment, n is 3 and R¹ is 5,6,7-triOCH₃.

In an embodiment, the compound is selected from:

or a pharmaceutically acceptable salt, solvate and/or prodrug thereof.

In another embodiment, the compound is:

In a further embodiment, the compound is:

The present disclosure also includes a pharmaceutical compositioncomprising one or more compounds of the present disclosure and apharmaceutically acceptable carrier.

The present disclosure also includes a method of treating cancercomprising administering one or more compounds of the present disclosureto a subject in need thereof.

In an embodiment, the cancer is breast cancer, leukemia, cervicalcancer, brain cancer, lung cancer, bladder cancer, kidney cancer,multiple myeloma or other blood cancers, colorectal cancer, CNS cancer,melanoma, ovarian cancer and prostate cancer. In another embodiment, thecancer comprises colchicine-resistant, paclitaxel-resistant,bortezomib-resistant, vinblastine-resistant and/or multidrug-resistanttumor cells.

In an embodiment, the one or more compounds of the present disclosureare administered in combination with one or more other anticanceragents. In another embodiment, the other anticancer agents are selectedfrom the group consisting of mitotic inhibitors, optionally paclitaxel;bcl2 family inhibitors, optionally ABT-737 and other inhibitors of theanti-apoptotic pathway; proteasome inhibitors, optionally bortezomib orcalfilzomib; signal transduction inhibitors, optionally gefitinib,erlotinib, dasatinib, imatinib or sunitinib; inhibitors of DNA repair,optionally iniparib, temozolomide or doxorubicin; and alkylating agents,optionally cyclophosphamide. In a further embodiment, the otheranticancer agent is paclitaxel.

In embodiments wherein the one or more compounds of the presentdisclosure are administered in combination with one or more otheranticancer agents, the dosage of the one or more compounds of thepresent disclosure is optionally less than the dosage of the one or morecompounds of the present disclosure when administered alone. In anotherembodiment, the dosage of the one or more compounds of the presentdisclosure is one half the dosage of the one or more compounds of thepresent disclosure when administered alone.

In embodiments wherein the one or more compounds of the presentdisclosure are administered in combination with one or more otheranticancer agents, the dosage of the other anticancer agent isoptionally less than the dosage of the other anticancer agent whenadministered alone. In another embodiment, the dosage of the otheranticancer agent is one half the dosage of the other anticancer agentwhen administered alone.

BRIEF DESCRIPTION OF THE DRAWINGS

The present disclosure will now be described in greater detail withreference to the drawings, in which:

FIG. 1 is a plot showing percentage cell death of asynchronously growingHeLa S3 cells treated with the compound CTR-20((E)-6-methoxy-3-(3-(2-methoxyphenyl)-3-oxoprop-1-enyl)quinolin-2(1H)-one) at concentrations of 0, 0.5, 1.0, 5.0 and 10.0 μMfor 24 or 72 hours (h).

FIG. 2 shows (A) flow cytometry profiles of HeLa cells at 72 hours (h)post-treatment with different concentrations (0, 0.75, 1.0, 3.0, 5.0,7.5 and 10.0 μM) of the compound CTR-17((E)-3-(3-(2-methoxyphenyl)-3-oxoprop-1-enyl) quinolin-2(1H)-one); and(B) cell cycle profiles at different time points (6, 12, 24, 48 and 72hours) after asynchronous HeLa cells were treated with 3.0 μM CTR-17(bottom row) in comparison to sham-treated HeLa cells (top row).

FIG. 3 shows flow cytometry profiles which were taken at different times(0, 6, 12, 24, 48 and 72 hours) after two breast cancer cell lines(MDA-MB-468 and MDA-MB-231; top and middle row, respectively) and onenon-cancer breast cell line (MCF-10A) were treated with 3.0 μM CTR-17(bottom row).

FIG. 4 shows flow cytometry profiles taken after asynchronous breastcancer cells (MDA-MB-231 and MCF-7; top and middle row, respectively)and their matching non-cancer breast cells (184B5; bottom row) weretreated with CTR-20 at 0.5 or 1 μM for 72 hours (h) in comparison tountreated cells.

FIG. 5 shows flow cytometry profiles which were taken after (A)MDA-MB-231 cells were treated with 1 μM CTR-20 for 4, 8, 24, 28 and 72hours in comparison to a sham control or (B) HeLa S3 cells were treatedwith 0.5, 1.0, or 2.5 μM CTR-20 for 24, 48 and 72 hours in comparison toa sham control.

FIG. 6 shows flow cytometry profiles that CTR-21 at >60 nMconcentrations caused mitotic arrest and, eventually, cell death. HeLacells were treated with different concentrations of CTR-21 for 6, 12,24, 48 and 72 hours (h) prior to analysis of their cell cycle profilesby flow cytometry.

FIG. 7 shows flow cytometry profiles that CTR-32 at >50 nMconcentrations caused mitotic arrest and, eventually, cell death. HeLacells were treated with different concentrations of CTR-32 for 6, 12,24, 48 and 72 hours (h) prior to analysis of their cell cycle profilesby flow cytometry.

FIG. 8 shows flow cytometry profiles that CTR-21 (30 nM) and CTR-32 (50nM) do not cause notable effects on the cell cycle progression of theMCF10A non-cancer cells, except a transient mitotic arrest at the12-hour time point (2^(nd) column from the left).

FIG. 9 illustrates that CTR-17, CTR-20, CTR-21 and CTR-32 preferentiallykill the fully malignant MCF10Ala breast cancer cells over thepre-malignant MCF10AT1 and the non-malignant MCF-10A breast cells. Cellswere treated with (A) CTR-17 at doses of 0.10, 0.39, 1.56 or 6.25 μM;(B) CTR-20 at doses of 0.10, 0.39, 1.56 or 6.25 μM; (C) CTR-21 at dosesof 7.81, 15.63, 31.25, 62.5, 125 or 250 nM; or (D) CTR-32 at doses of7.81, 15.63, 31.25, 62.5, 125 or 250 nM for 72 hours. Cell viability wasdetermined using an SRB assay.

FIG. 10 shows exemplary images of (A) HeLa cells that were sham-treatedor treated with 3 μM CTR-17 and stained with an antibody specific forγ-tubulin (far left), stained with an antibody specific for α-tubulin(second from left), counterstained DNA with DAPI (second from right) andmerged images (far right). Scale bar denotes 10 μm; (B) of a highermagnification of the DAPI and merged images of HeLa cells that weresham-treated or treated with 3 μM CTR-17; and (C) of HEK293T, MDA-MB-468and MDA-MB-231 cells that were treated with 3 μM CTR-17 and stained withan antibody specific for γ-tubulin (far left), stained with an antibodyspecific for α-tubulin (second from left), counterstained DNA with DAPI(second from right) and merged images (far right). Scale bar denotes 2μm for images in top and middle row and 5 μm for images in bottom row.White arrows in (B) and (C) denote the failure of proper alignment atthe centre plate or uneven segregation of chromosomes.

FIG. 11 is a plot showing the percentage of mitotic cells for non-cancercells MCF-10A and 184B5 and cancer cells MDA-MB-231, HeLa, MDA-MB-468and HEK293T that were either sham-treated or treated with 3.0 μM CTR-17for 12 hours (h) or 24 hours, followed by analyses of cell cycleprogression, centrosome abnormalities, and chromosomealignment/segregation.

FIG. 12 shows exemplary images of (A) approximately metaphase and (B)approximately anaphase and telophase/cytokinesis for asynchronouslygrowing MCF-7, MDA-MB-231 and HeLa S3 cells treated with sham (MCF-7only) or 1 μM CTR-20 for 24 hours (h). Cells were then collected, fixedwith methanol, and incubated with an antibody specific for α-tubulin(far left column of FIGS. 12A and 12B) and then counterstained DNA withDRAQ5 (second from the left column of FIGS. 12A and 12B) and mergedimages (far right column of FIG. 12A; second from right and far rightcolumn of FIG. 12B). The internal box in the telophase/cytokinesis MCF-7sample in FIG. 12B shows uneven cell division. Scale bars on all imagesexcept for the internal box denote 5 μm.

FIG. 13 is a plot showing the percentage of mitotic cells for non-cancercells MCF-10A and 184B5 compared to cancer cells MDA-MB-231, HeLa andMCF-7 sham treated or treated with 1 μM CTR-20 for 24 hours.

FIG. 14 shows plots of the percentage of cells in various cell stages(from left to right on each of FIGS. 14A and 14B: prometaphase,metaphase, anaphase/telophase and cytokinesis) for HeLa S3 cells growingon cover slips synchronized by double thymidine treatment then releasedinto fresh medium either in the absence (A; sham treated) or (B)presence of 1.0 μM CTR-20 for a duration of 7.5, 8.5, 9.5, 10, 10.5 or11.5 hours.

FIG. 15 shows that chromosomes in HeLa cells are misaligned in thepresence of CTR-21 (middle row) and CTR-32 (bottom row). HeLa cells weretreated with 30 nM CTR-21 or 50 nM CTR-32 for 12 hours prior to fixingin methanol and immunostaining with an antibody specific for γ-tubulin(green; first column) or α-tubulin (red; second column), which were thencounterstained with Draq5 (blue; third, fifth and seventh columns).Merged images are shown on the forth, sixth and eighth columns from theleft. Failure to align properly at the center plate and perturbedseparation of chromosomes are shown by white arrows.

FIG. 16 shows that CTR-21 and CTR-32 activate Bcl-X_(L) in the cellsarrested at mitosis. HeLa cells were treated with CTR-21 (15 or 30 nM)or CTR-32 (30 or 50 nM) for 6, 12 or 24 hours (h). Whole cell lysateswere collected at the scheduled time points and used to perform SDS PAGEprotein separation, followed by Western blotting with antibodiesspecific for proteins listed on the right of the panel. p-S62 Bcl-X_(L)denotes Bcl-X_(L) phosphorylated on the serine 62 residue (i.e.,Bcl-X_(L) is activated). GAPDH was used as the loading control. Itshould be noted that the levels of cyclin B and the high molecularweight (i.e., phosphorylated) of Cdc25C are much higher in the presenceof CTR-21 or CTR-31 than in the absence either of the compounds.

FIG. 17 shows (A) exemplary HeLa cell cycle histograms after treatmentwith CTR-17 (3 μM; second from right) or CTR-20 (1 μM; far right) for 12hours (which is defined as time 0 post-release) in comparison tountreated (far left) and sham treatment (second from left); and (B)exemplary cell cycle histograms of cells treated with CTR-17 (top) andCTR-20 (bottom) as described for FIG. 17A then washed twice with 1×PBSand re-suspended in 10 ml of pre-warmed, drug-free medium for durationsof 3, 6, 9 or 12 hours (from left to right).

FIG. 18 shows that the effects of CTR-21 and CTR-32 are reversible. (A)HeLa cells arrested at G2/M phase by treating them with CTR-21 (30 nM)or CTR-32 (50 nM) for 12 hours (which is designated as time 0 h) werewashed twice with 1×PBS and then released into cell cycle by culturingin drug-free medium for 1, 2, 4, 6 and 8 hours (h). The cell cycleprogression was then analysed by flow cytometry (lower panels). (B)Exemplary images of HeLa cells at 1, 2, 4, 6 and 8 hours post releasefrom the CTR treatment as shown in FIG. 18A.

FIG. 19 shows the results of Western blotting carried out with ananti-PARP antibody at time points (hours, h) of 12, 24 and 48 hoursusing whole cell extracts prepared from asynchronous HeLa cells (left)and 184B5 cells (right) treated with sham or 3 μM of CTR-17 (top image).GAPDH was used as a loading control (bottom image).

FIG. 20 shows (A) exemplary images of synchronous HeLa cells treatedwith CTR-17 (3.0 μM) for 24 hours, then fed with EdU (10.0 μM) for 1hour immediately prior to harvesting them for analysis (bottom row) incomparison to a sham control (top row); and (B) exemplary images of cellimmunostaining with an antibody specific for γ-H2AX (second column fromleft) to detect damaged DNA (i.e., damage repairing) of CTR-17 treatedHeLa cells (bottom row) in comparison to a sham control (top row).Etoposide (50.0 μM) was used as a positive control (middle row). Scalebar denotes 20 μM.

FIG. 21 shows images of Western blots carried out with whole cellextracts prepared from asynchronously growing HeLa cells. Equal amountsof proteins were resolved by SDS-PAGE, and blotting was carried out withantibodies specific for those proteins listed at left of the gels (fromtop to bottom: p-Cdk1, Y15; pCdk1, T161; Cdk1; Cyclin B; Wee1; Cdc25C;p-Cdc25C, S216; pCdc25C, T48). Time points in hours (h) arepost-treatment with 3.0 μM CTR-17 (right 4 columns) or sham control(left 5 columns). GAPDH (bottom image) was used as a loading control.“p-” denotes phosphorylation.

FIG. 22 shows flow cytometry profiles of HeLa cells synchronised at theG1/S border by double thymidine (DT) block then released into the cellcycle in the absence (sham; top row) or presence of CTR-17 (3.0 μM;bottom row) for a duration of 3, 6, 9, 12, 16 or 48 hours (h) incomparison to controls.

FIG. 23 shows the results of analysis of HeLa cells synchronized at theG1/S border by double thymidine (DT) block then released into completemedium at time 0 in the absence (sham; FIG. 23A) or presence (FIG. 23B)of 3.0 μM CTR-17 for a duration of 1, 3, 6, 9, 12, 14, 16, 18 or 20hours (from left to right). Equal amounts of proteins were resolved bySDS-PAGE, followed by Western blotting with antibodies specific for theproteins, from top to bottom: p-Cdk1, Y15; pCdk1, T161; Cdk1; p-Cdc25C,T48; Cdc25C; securin; cyclin B; cyclin E; cyclin A; p-histone H3;histone H3; GAPDH (loading control); BubR1; and GAPDH (loading control).“p-” denotes phosphoprotein.

FIG. 24 shows the Western blot results of HeLa cells synchronised at theG1/S boundary by double thymidine (DT) block then sham treated (leftthree columns), treated with 20 ng/ml nocodazole (middle three columns),or treated with 3.0 μM CTR-17 (right three columns) for durations of 6,9 and 12 hours (h). Total protein extracts were subjected toimmunoprecipitation with an anti-BubR1 antibody, followed by proteinseparation by SDS-PAGE and Western blotting with an anti-Cdc20 antibodyto examine the interaction between BubR1 and Cdc20.

FIG. 25 shows exemplary images of asynchronously growing HeLa cellssham-treated (top two rows) or treated with CTR-17 (3.0 μM; bottom tworows) for 12 hours, fixed, and then immunostained with antibodiesspecific for BubR1 (far left column) or Cenp-B (second from left column)(centromere staining). The column second from the right shows mergedimages and the far right column shows bright field images. The scale bardenotes 5 μm for the top row and second row from the bottom and denotes2 μm for the other images.

FIG. 26 is a plot of absorbance at 340 nm as a function of time (min)after purified porcine tubulin and 1.0 mM GTP were added to a reactionmixture containing 10.0 μM paclitaxel, 3.0 μM CTR-17, 1.0 μM CTR-20, or5.0 μM nocodazole then polymerization of tubulin was monitored everyminute for one hour at 340 nm and 37° C. by spectrophotometry.

FIG. 27 shows that CTR-21 and CTR-32 effectively inhibit microtubulepolymerization. Paclitaxel, CTR-20, CTR-21, CTR-32 or colchicine wasadded to a reaction mixture containing highly purified porcine tubulinand 1.0 mM GTP. The reaction was carried out for 1 hour at 37° C., whilemonitoring fluorescence emission at 1-minute intervals. The fluorescenceexcitation was at 350 nm and the emission was recorded at 430 nm.

FIG. 28 shows (A) the results of HeLa cells that were sham-treated(Sham), treated with 50.0 nM paclitaxel (Tax), 50.0 ng/ml nocodazole(Noc), 3.0 μM CTR-17, or 1.0 μM CTR-20 for 12 hours then the celllysates were separated into polymerization (Pol) and soluble (Sol)fractions, and equal amounts of proteins resolved by SDS-PAGE, followedby immunoblotting with an antibody specific for α-tubulin (upper panel).Bands were quantified with densitometry and expressed in a graph form(lower panel). (B) HeLa cells treated with different concentrations ofCTR-17 or CTR-20 and subjected to fractionation and immunoblotting asdescribed for FIG. 28A.

FIG. 29 shows plots demonstrating that CTR-17 (A) and CTR-20 (B)quenched the intrinsic tryptophan fluorescence of tubulin in adose-dependent manner. Purified tubulin dissolved in 25 mM PIPES bufferwas incubated in the presence or absence of different concentrations ofCTR compounds for 30 minutes at 37° C. Fluorescence was monitored byexcitation of the reaction mixture at 295 nm, and the emission spectrawere recorded from 315 to 370 nm; and plots of the change influorescence intensity as a function of drug concentrations of CTR-17(C) and CTR-20 (D) to determine the dissociation constant. ΔF is thechange in fluorescence intensity of tubulin when bound by the CTRcompounds. Data are an average of five independent experiments.

FIG. 30 shows results suggesting that (A) CTR-17 and CTR-20, similar tocolchicine, did not bind to the vinblastine binding site on the tubulin.25 μM each of colchicine, CTR-17, CTR-20, or vinblastine was incubatedwith tubulin for 1 hour to promote the formation of complexes betweentubulin and each of these compounds. The resultant complexes wereincubated for 30 minutes with 5 μM of the fluorescent BODIPYFL-vinblastine to determine if the binding of each compound to tubulinis in competition with vinblastine. (B) CTR-17 binds to tubulin at ornear the colchicine-binding site. The tubulin-fluorescent colchicinecomplex was incubated with increasing concentrations of eithervinblastine or CTR-17. CTR-17 but not vinblastine competed with(fluorescent) colchicine. CTR-17 (C) and CTR-20 (D) depressed thefluorescence of the colchicine-tubulin complex in a dose-dependentmanner. Tubulin was incubated with different concentrations of CTR-17 orCTR-20 for 1 hour, in three separate sets with concentrations ofcolchicine of 3.0, 5.0 and 8.0 μM (for CTR-17) or 1.0, 3.0 and 5.0 μM(for CTR-20). Inhibitory constants of CTR-17 (E) and CTR-20 (F). Thefluorescence intensity of the final tubulin complex (FIGS. 30C and 30D)was used to determine the inhibitory concentration (Ki) utilizing amodified Dixon plot. F is the fluorescence of the complexes of CTR-17(or CTR-20)-colchicine-tubulin or vinblastine-colchicine-tubulincomplex, and F0 is the fluorescence of the colchicine-tubulin complex.Data are an average of at least four independent experiments.

FIG. 31 shows images of (A) the results of molecular docking predictingthe tubulin-binding sites of colchicine, CTR-17, CTR-20, podophyllotoxinand vinblastine using the 3D X-ray structure of tubulin (PDB code:1SA0); and (B) the chemical structures of colchicine, CTR-17, CTR-20 andpodophyllotoxin.

FIG. 32 shows images of the predicted interaction between the tubulinheterodimer (PDB code: 1SA0) and colchicine (A), CTR-20 (B), or CTR-17(C) in a 3D pattern. 2D ligand interaction diagrams show potentialchemical interactions between amino acids and compounds within adistance of 4 Å to colchicine (A′), CTR-20 (B′), or CTR-17 (C′).

FIG. 33 shows images of (A) Western blotting of whole cell extractsprepared from the parental KB-3-1 and MDR1-overexpressing KB-C-2isogenic cell lines; and (B) Western blotting of whole cell extractsprepared from the parental H69 and MRP1-overexpressing H69-AR isogeniccell lines.

FIG. 34 shows (A) part of the CTR-17 data presented in Table 8 in agraph form; and (B) part of the CTR-20 data presented in Table 8 in agraph form. CI denotes combination index. CI<1.0, CI=1.0 and CI>1.0 aresynergistic, additive and antagonistic, respectively (Chou, 2006). Datapresented are mean±S.E.M value of triplicates of at least fourindependent experiments.

FIG. 35 shows that CTR compounds kill multi-drug resistant and sensitivecells with similar efficacy. (A) The multidrug-resistant MDA-MB231TaxRcells express high levels of P-glycoprotein (P-gp; MDR1). Whole cellextracts of MDA-MB231 cells selected at different concentrations ofpaclitaxel (2.0, 10.0, 15.0, 30.0 and 100.0 nM) along with the parentalMDA-MB231 (WT) were subjected to SDS-PAGE and Western blotting with ananti-MDR1 antibody. GAPDH was used as a loading control. (B)MDR1-overexpressing MDA-MB231TaxR (selected at 100 nM paclitaxel) andits parental MDA-MB231 cells were killed by CTR compounds with similarefficacy, while the MDA-MB231TaxR is over 114-fold more resistant topaclitaxel and at least 15-fold more resistant to vinblastine than theMDA-MB231.

FIG. 36 shows that CTR-20, CTR-21 and CTR-32 kill bortezomib-resistantRPMI-8226 cells (RPMI-8226BTZR) with similar potency to the parentalRPMI-8226 multiple myeloma cells.

FIG. 37 shows that CTR-20, CTR-21 and CTR-32 are synergistic in killingthe multidrug-resistant MDA-MB231TaxR (selected in 100 nM) cells whenused in combination with paclitaxel. (A) Synergistic effects of CTR-20in combination with paclitaxel against MDA-MB231TaxR. Lanes denote: 300nM paclitaxel (Tax) (lane 1), 312.5 nM CTR-20 (lanes 2, 5, 8 & 11),312.5 nM CTR-20 plus 300 nM paclitaxel (lane 3), 150 nM paclitaxel (lane4), 312.5 nM CTR-20 plus 150 nM paclitaxel (lane 6), 75 nM paclitaxel(lane 7), 312.5 nM CTR-20 plus 75 nM paclitaxel (lane 9), 37.5 nMpaclitaxel (lane 10), and 312.5 nM CTR-20 plus 37.5 nM paclitaxel (lane12). (B) Synergistic effects of CTR-21 in combination with paclitaxel(Tax) against MDA-MB231TaxR (selected in 100 nM). Lanes denote: 300 nMpaclitaxel (Tax) (lane 1), 23 nM CTR-21 (lanes 2, 5, 8, 11 & 14), 23 nMCTR-21 plus 300 nM paclitaxel (lane 3), 150 nM paclitaxel (lane 4), 23nM CTR-21 plus 150 nM paclitaxel (lane 6), 75 nM paclitaxel (lane 7), 23nM CTR-21 plus 75 nM paclitaxel (lane 9), 37.5 nM paclitaxel (lane 10),23 nM CTR-21 plus 37.5 nM paclitaxel (lane 12) and 18.75 nM paclitaxel(lane 13), 23 nM CTR-21 plus 18.75 nM paclitaxel (lane 15). (C)Synergistic effects of CTR-32 in combination with paclitaxel againstMDA-MB231TaxR (selected in 100 nM). Lanes denote: 300 nM paclitaxel(lane 1), 23 nM CTR-32 (lanes 2, 5, 8, 11 & 14), 23 nM CTR-32 plus 300nM paclitaxel (lane 3), 150 nM paclitaxel (lane 4), 23 nM CTR-32 plus150 nM paclitaxel (lane 6), 75 nM paclitaxel (lane 7), 23 nM CTR-32 plus75 nM paclitaxel (lane 9), 37.5 nM paclitaxel (lane 10), 23 nM CTR-32plus 37.5 nM paclitaxel (lane 12) and 18.75 nM paclitaxel (lane 13), 23nM CTR-32 plus 18.75 nM paclitaxel (lane 15). “CI” denotes combinationalindex: CI<1.0, CI=1.0 and CI>1.0 are synergistic, additive andantagonistic, respectively. Data presented are mean±S.E.M value oftriplicates of at least three independent experiments.

FIG. 38 shows that CTR-20 is synergistic when used in combination withABT-737 against MDA-MB231 cells. (A) Lanes denote: 6.25 μM ABT-737(lanes 1, 4 & 7), 0.4 μM CTR-20 (lane 2), 0.4 μM CTR-20 plus 6.25 μMABT-737 (lane 3), 0.2 μM CTR-20 (lane 5), 0.2 μM CTR-20 plus 6.25 μMABT-737 (lane 6), 0.1 μM CTR-20 (lane 8) and 0.1 μM CTR-20 plus 6.25 μMABT-737 (lane 9). (B) Lanes denote: 3.125 μM ABT-737 (lanes 1, 4 & 7),0.4 μM CTR-20 (lane 2), 0.4 μM CTR-20 plus 3.125 μM ABT-737 (lane 3),0.2 μM CTR-20 (lane 5), 0.2 μM CTR-20 plus 3.125 μM ABT-737 (lane 6),0.1 μM CTR-20 (lane 8) and 0.1 μM CTR-20 plus 3.125 μM ABT-737 (lane 9).CI denotes combinational index. Data presented are mean±S.E.M value oftriplicates of at least three independent experiments.

FIG. 39 shows flow cytometry profiles of CTR-20, ABT-737 and thecombination of the two against MDA-MB231. MDA-MB231 cells were shamtreated (Sham) or treated with 6.25 μM ABT-737, 3.13 μM ABT-737, 0.4 μMCTR-20 (CTR), 0.4 μM CTR-20 plus 6.25 μM ABT-737 or 0.4 μM CTR-20 plus3.13 μM ABT-737 for 6, 12, 24, 48 or 72 hours (h). Note that thecombination of 0.4 μM CTR-20 plus 6.25 μM ABT-737 completely killedMDA-MB231 by 72 hours of treatment.

FIG. 40 shows Western blot data indicating that CTR-20 in combinationwith ABT-737 may kill cells through the Bcl2 apoptotic pathways. Westernblotting carried out with whole cell extracts prepared from MDA-MB231cells treated with CTR-20, ABT-737 or in combination of the two for 12hours. Immunostaining was carried out with antibodies specific for theproteins listed on the right of the blots. GAPDH was used as a loadingcontrol. “p-” denotes phosphorylation.

FIG. 41 shows summary of data obtained from screening the NCI-60 cancerpanel. Ten μM of CTR-20 was used to examine the drug's efficacy againstthe NCI-60 cancer cell lines including: six leukemia cell lines, ninenon-small cell lung cancer cell lines, seven colorectal cancer celllines, six CNS cancer cell lines, nine melanoma cell lines, sevenovarian cancer cell lines, seven renal cancer cell lines, two prostatecancer cell lines and six breast cancer cell lines. Screening method wascarried out by a sulforhodamine B (SRB) colorimetric assay.

FIG. 42 shows (A) a plot of tumor size (volume in mm³) as a function ofdays post-treatment “D” in response to drug treatments, alone or incombination with paclitaxel; and (B) exemplary images of representativeATH490 athymic mice engrafted with MDA-MB-231 human metastatic breastcancer cells that were treated with vehicle only (top row) or treatedwith the drugs paclitaxel (Tax; second row from top); CTR-17 (third rowfrom top); CTR-20 (third row from bottom); paclitaxel and CTR-17 (secondrow from bottom); and paclitaxel and CTR-20 (bottom row). Numbers inbrackets are mg/kg body weight.

FIG. 43 is a plot showing normalized body weight of six-week old ATH40athymic nude mice treated with vehicle, paclitaxel (Tax), CTR-17,CTR-20, paclitaxel and CTR-17 or paclitaxel and CTR-20 as a function ofdays (0, 2, 6, 14, 20, 24, 27 or 30) post-drug treatment. The numbers inbrackets show drug concentrations in mg/kg body weight. The body weightsof ATH490 mice were normalized based on the body weight on day 0 (100%).

FIG. 44 shows plots of the weights of four different organs (liver (A),spleen (B), kidney (C) and lung (D)) of ATH490 mice from differenttreatments measured at 30-day post-treatment. All values are presentedas mean±S.E.M. Each organ weight (%) was normalized with total bodyweight.

FIG. 45 shows (A) images of livers of ATH490 athymic mice that weretreated with sham (top left), 10 mg/kg paclitaxel (top right), 30 mg/kgCTR-17 (middle left), 30 mg/kg CTR-20 (middle right), 5 mg/kg paclitaxelplus 15 mg/kg CTR-17 (bottom left) and 5 mg/kg paclitaxel plus 15 mg/kgCTR-20 (bottom right). White arrows indicate mitotic cells. (B) is aplot showing the number of mitotic cells/mm² as a function of thetreatment regimens of (A).

FIG. 46 shows images of spleens of ATH490 athymic mice that weresham-treated (vehicle only; top left) or treated with 10 mg/kgpaclitaxel (top right), 30 mg/kg CTR-17 (middle left), 30 mg/kg CTR-20(middle right), 5 mg/kg paclitaxel plus 15 mg/kg CTR-17 (bottom left)and 5 mg/kg paclitaxel plus 15 mg/kg CTR-20 (bottom right) for 30 days,followed by toxicity analysis after spleen tissues were H & E stained.Arrows indicate the presence of macrophages in the red pulp (RP). Imageswere taken using a Zeiss EPI-fluorescent microscope (10× objective).

FIG. 47 shows images of kidneys of ATH490 mice that were sham-treated(vehicle only; top left) or treated with 10 mg/kg paclitaxel (topright), 30 mg/kg CTR-17 (middle left), 30 mg/kg CTR-20 (middle right), 5mg/kg paclitaxel plus 15 mg/kg CTR-17 (bottom left) and 5 mg/kgpaclitaxel plus 15 mg/kg CTR-20 (bottom right). At day 30, kidneys wereharvested, stained with H&E, and observed under a Zeiss EPI-fluorescentmicroscope (40× objective). Arrows pointing to the top right imageindicate hyaline.

DETAILED DESCRIPTION I. Definitions

Unless otherwise indicated, the definitions and embodiments described inthis and other sections are intended to be applicable to all embodimentsand aspects of the present disclosure herein described for which theyare suitable as would be understood by a person skilled in the art.

In embodiments of the disclosure, the compounds described herein have atleast one asymmetric center. Where compounds possess more than oneasymmetric center, they may exist as diastereomers. It is to beunderstood that all such isomers and mixtures thereof in any proportionare encompassed within the scope of the present disclosure. It is to befurther understood that while the stereochemistry of the compounds maybe as shown in any given compound listed herein, such compounds may alsocontain certain amounts (e.g. less than 20%, optionally less than 10%,optionally less than 5%, optionally less than 3%) of the correspondingcompound having alternate stereochemistry.

In embodiments of the disclosure, the compounds described herein have atleast one double bond capable of geometric isomerism; for example, thecompound may exist as a cis or a trans isomer. It is to be understoodthat all such isomers and mixtures thereof in any proportion areencompassed within the scope of the present disclosure. It is to befurther understood that while the isomerism of the compounds may be asshown in any given compound listed herein, such compounds may alsocontain certain amounts (e.g. less than 20%, optionally less than 10%,optionally less than 5%, optionally less than 3%) of the correspondingcompound having alternate isomerism.

The term “alkyl” as used herein, whether it is used alone or as part ofanother group, means straight or branched chain, saturated alkyl groups.The number of carbon atoms that are possible in the referenced alkylgroup are indicated by the numerical prefix “C_(n1-n2)”. For example,the term C₁₋₆alkyl means an alkyl group having 1, 2, 3, 4, 5 or 6 carbonatoms.

The term “alkenyl” as used herein, whether it is used alone or as partof another group, means straight or branched chain, unsaturated alkenylgroups. The number of carbon atoms that are possible in the referencedalkenyl group are indicated by the numerical prefix “C_(n1-n2)”. Forexample, the term C₂₋₆alkenyl means an alkenyl group having 2, 3, 4, 5or 6 carbon atoms and at least one double bond.

The term “halo” as used herein refers to a halogen atom and includes F,Cl and Br.

The term “haloalkyl” as used herein refers to an alkyl group wherein oneor more, including all of the available hydrogen atoms are replaced by ahalogen atom. The number of carbon atoms that are possible in thereferenced haloalkyl group are indicated by the numerical prefix“C_(n1-n2)”. For example, the term C₁₋₆haloalkyl means a haloalkyl grouphaving 1, 2, 3, 4, 5 or 6 carbon atoms. In an embodiment, the halogen isa fluorine, in which case the haloalkyl is optionally referred to hereinas a “fluoroalkyl” group. It is an embodiment that all of the hydrogenatoms are replaced by fluorine atoms. For example, the haloalkyl groupcan be trifluoromethyl, pentafluoroethyl and the like. It is anembodiment of the present disclosure that the haloalkyl group istrifluoromethyl.

The term “subject” as used herein includes all members of the animalkingdom including mammals, and optionally refers to humans.

The term “pharmaceutically acceptable” means compatible with thetreatment of subjects, for example, mammals such as humans.

The term “pharmaceutically acceptable salt” as used herein means an acidaddition salt that is compatible with the treatment of subjects.

An “acid addition salt that is compatible with the treatment ofsubjects” is any non-toxic organic or inorganic salt of any basiccompound. Basic compounds that form an acid addition salt include, forexample, compounds comprising an amine group susceptible to protonation.Illustrative inorganic acids which form suitable salts includehydrochloric, hydrobromic, sulfuric and phosphoric acids, as well asmetal salts such as sodium monohydrogen orthophosphate and potassiumhydrogen sulfate. Illustrative organic acids that form suitable saltsinclude mono-, di-, and tricarboxylic acids such as glycolic, lactic,pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric,ascorbic, maleic, benzoic, phenylacetic, cinnamic and salicylic acids,as well as sulfonic acids such as p-toluene sulfonic and methanesulfonicacids. Such salts may exist in a hydrated, solvated or substantiallyanhydrous form. In general, acid addition salts are more soluble inwater and various hydrophilic organic solvents, and generallydemonstrate higher melting points in comparison to their free baseforms. The selection of a suitable salt can be made by a person skilledin the art. The formation of a desired acid addition salt is, forexample, achieved using standard techniques. For example, the neutralcompound is treated with the desired acid in a suitable solvent and thesalt which is thereby formed then isolated by filtration, extractionand/or any other suitable method.

The term “solvates” as used herein in reference to a compound refers tocomplexes formed between the compound and a solvent from which thecompound is precipitated or in which the compound is made. Accordingly,the term “solvate” as used herein means a compound, or a salt of acompound, wherein molecules of a suitable solvent are incorporated inthe crystal lattice. Examples of suitable solvents are ethanol, waterand the like. When water is the solvent, the molecule is optionallyreferred to as a “hydrate”. The formation of solvates will varydepending on the compound and the solvate. In general, solvates areformed by dissolving the compound in an appropriate solvent andisolating the solvate by cooling or using an antisolvent. The solvate istypically dried or azeotroped under ambient conditions. The selection ofsuitable conditions to form a particular solvate can be made by a personskilled in the art.

The term “prodrug” as used herein in reference to a compound refers to aderivative of the compound that reacts under biological conditions toprovide the compound. In an embodiment, the prodrug comprises aconventional ester formed with an available amino group. For example, anavailable amino group is acylated using an activated acid in thepresence of a base, and optionally, in an inert solvent (e.g. an acidchloride in pyridine). Some common esters which have been used asprodrugs are phenyl esters, aliphatic (C₁-C₂₄) esters, acyloxymethylesters, carbamates and amino acid esters.

The one or more compounds of the application are, for example,administered to the subject or used in an “effective amount”.

As used herein, the term “effective amount” and the like means an amounteffective, at dosages and for periods of time necessary to achieve adesired result. For example, in the context of treating cancer, aneffective amount of the one or more compounds of the disclosure is anamount that, for example, reduces the cancer compared to the cancerwithout administration of the one or more compounds of the disclosure.Effective amounts may vary according to factors such as the diseasestate, age, sex, weight and/or species of the subject. The amount of agiven compound that will correspond to such an amount will varydepending upon various factors, such as the given compound, thepharmaceutical formulation, the route of administration, the type ofcondition, disease or disorder being treated, the identity of thesubject being treated, and the like, but can nevertheless be routinelydetermined by one skilled in the art.

II. Compounds and Methods of Preparation Thereof

The microtubule is the target for several different anticancertherapeutic agents including colchicine and paclitaxel. However, the useof colchicine as an anticancer agent has not been approved, for example,by the U.S. Food and Drug Administration due mainly to its inherenttoxicity. Accordingly, it is an object of the studies of the presentdisclosure to develop an anticancer agent targeting thecolchicine-binding site on microtubule with minimum toxicity. Severalchalcone derivatives were synthesized and examined. Data from thepresent study with three human breast cancer cell lines (MDA-MB-468,MDA-MB-231 and MCF-7) and two matching non-cancer breast cell lines(184B5 and MCF-10A) showed that CTR-17 and CTR-20 are useful anticancerleads. The study was also expanded to several other cancer cell linesincluding K562 (chronic myelogenous leukemia [CML] cell line), HeLa(cervical cancer), U87MG (brain cancer), T98G (temozolomide-resistantbrain cancer), NCI-H1975 (lung cancer), A549 (lung cancer), RPMI-8226(multiple myeloma), RPMI-8226-BR (Bortezomib-resistant RPMI-8226 cellline for the compound CTR-20 only), KB-3-1 (cervical cancer), KB-C-2(colchicine-resistant and paclitaxel-resistant KB-3-1 cell line),ANBL6-BR (bortezomib-resistant multiple myeloma for the compound CTR-20only), H69 (small cell lung cancer) and H69AR (multidrug-resistant smallcell lung epithelial cancer). It was found that: (a) CTR-17 and CTR-20preferentially kill cancer over non-cancer cells, up to 26 times(CTR-17, on MDA-MB-468 versus MCF-10A) and 24 times (CTR-20, on HeLaversus MCF-10A); (b) CTR-17 and CTR-20 induce a prolonged cell cyclearrest at the spindle checkpoint step in a cancer cell-specific manner,eventually leading to cancer cell death by apoptosis; (c) CTR-17 andCTR-20 inhibit tubulin polymerisation; (d) the dissociation constants ofCTR-17 and CTR-20 are 4.58±0.95 μM and 5.09±0.49 μM, respectively; (e)the microtubule binding sites of both CTR-17 and CTR-20 almost overlapwith that of colchicine; (f) unlike colchicine, the effects of theCTR-17 and CTR-20 compounds are reversible; (g) data from in silicomolecular docking studies suggests, while not wishing to be limited bytheory, that CTR-17, CTR-20 and colchicine, respectively, form one, twoand three hydrogen bonds (H-bonds) with amino acid residues of tubulin,in addition to forming strong Van der Waals interactions; (h) CTR-17 andCTR-20 kill MDR1-overexpressing and MRP1-overexpressingmultidrug-resistant cancer cells (which are alsopaclitaxel/colchicine-resistant); (i) CTR-20 killed bortezomib-resistantmultiple myeloma cells (IC₅₀ values of RPMI-8226-BR and ANBL6-BR were0.28±0.03 and 0.76±0.28 μM, respectively); (j) the combinationaltreatment of MDR1 overexpressing KB-C-2 cells with paclitaxel and CTR-17or CTR-20 showed synergistic effects; (k) data from in vitro and animalstudies shows that both CTR-17 and CTR-20 are useful as antitumoragents; and (l) data from studies with engrafted mice showed that thecombination of ½ doses of CTR-20 and paclitaxel is more efficient thanthe full dose of either compound alone, without causing any notableill-effects. Together, the data indicates that CTR compounds, forexample, CTR-20, in one embodiment, are useful anticancer agents thatcan, for example, kill many different cancer cells includingcolchicine/paclitaxel-resistant, bortezomib-resistant, andmultidrug-resistant tumor cells with no notable ill-effects which wereobserved in the present studies on non-cancer cells and normal mouseorgans. Further, (m) Studies carried out with 16 compounds (CTR-21 toCTR-40) shows that they kill tumor cells with IC₅₀ values ranging from5.34 nM (CTR-21 against RPMI-8226) to 2.69 μM (CTR-27 againstMDA-MB231); (n) further studies showed that CTR-21 and CTR-32 are potentagainst tumor cells, MDA-MB231, MCF-7, HeLa and RPMI-8226, as their IC₅₀values are in the nonomolar range; (o) a study with isogenic breast andbreast cancer cell lines showed that CTR-17, CTR-20, CTR-21 and CTR-32preferentially kill fully malignant cells over pre-cancer or non-cancercells; (p) CTR-21 and CTR-32 are microtubule polymerization inhibitors;(q) similar to CTR-20, CTR-21 and CTR-32 are reversible mitoticinhibitors; (r) the combination of CTR-20 and ABT-737, an inhibitor ofthe Bcl2 anti-apoptotic family proteins, is synergistic againstMDA-MB231 triple-negative metastatic breast cancer, as the combinationalindex is 0.07-0.10; (s) CTR-20, CTR-21 and CTR-32 bortezomib-resistantRPMI-8226BTZR cells; (t) CTR-20, CTR-21 and CTR-32 kill the multidrug-and paclitaxel-resistant MDA-MB231TaxR cells, and also when combinedwith paclitaxel and CTR-20; (u) CTR-20 kills NCI-60 cancer cell linesincluding: six leukemia cell lines, nine non-small cell lung cancer celllines, seven colorectal cancer cell lines, six CNS cancer cell lines,nine melanoma cell lines, seven ovarian cancer cell lines, seven renalcancer cell lines, two prostate cancer cell lines and six breast cancercell lines.

Accordingly, the present disclosure includes a compound of Formula I:

wherein

A is O or S;

n is 0, 1, 2 or 3;

when n is 1, R¹ is halo, C₁₋₆alkyl, C₂₋₆alkenyl or —X—C₁₋₆alkyl;

when n is 2 or 3, each R¹ is independently halo, C₁₋₆alkyl, C₂₋₆alkenylor —X—C₁₋₆alkyl; or two R¹ together form a methylenedioxy group that isattached to two adjacent ring carbon atoms;

R² is C₁₋₆alkyl or C₁₋₆haloalkyl;

R³ is absent or is halo, —X—C₁₋₆alkyl or —X—C₁₋₆haloalkyl; and

each X is independently O or S,

or a pharmaceutically acceptable salt, solvate and/or prodrug thereof.

In an embodiment, A is O. In another embodiment, X is S.

In an embodiment, X is O. In another embodiment, X is S.

In an embodiment, R³ is absent. In another embodiment, R³ is F,—X—C₁₋₄alkyl or —X—C₁₋₄haloalkyl. In a further embodiment, R³ is F,—O—C₁₋₄alkyl or —O—C₁₋₄haloalkyl. It is an embodiment that R³ is F,—OCH₃ or —OCF₃. In another embodiment, R³ is 4′-OCH₃, 5′-OCH₃, 6′-OCH₃,4′-OCF₃, 4′-F or 5′-F.

In an embodiment, R² is C₁₋₄alkyl or C₁₋₄haloalkyl. In anotherembodiment, R² is CH₃ or CF₃. In a further embodiment, R² is CH₃. It isan embodiment of the present disclosure that R² is CF₃.

In an embodiment, n is 0, 1 or 2. In another embodiment, n is 0 or 1. Ina further embodiment, n is 0. It is an embodiment that n is 1. Inanother embodiment, n is 2. In a further embodiment, n is 3.

In an embodiment, n is 1 and R¹ is halo, C₁₋₄alkyl, C₂₋₄alkenyl or—X—C₁₋₄alkyl. In another embodiment, n is 1 and R¹ is CH₃ or OCH₃. In afurther embodiment, n is 1 and R¹ is 6-OCH₃, 7-OCH₃, 8-OCH₃, 6-OC₂H₅,6-SCH₃, 7-SCH₃, 6-CH₃, 6-C₂H₅, 6-F, 6-Cl, 6-Br, 7-F, 7-Cl or 7-Br. It isan embodiment that n is 1 and R¹ is 6-CH₃, 6-OCH₃ or 7-OCH₃. In anotherembodiment, n is 1 and R¹ is 6-OCH₃.

In an embodiment, n is 2 and each R¹ is independently halo, C₁₋₄alkyl,C₂₋₄alkenyl or —X—C₁₋₄alkyl; or two R¹ together form a methylenedioxygroup that is attached to two adjacent ring carbon atoms. In anotherembodiment, each R¹ is independently CH₃ or OCH₃; or two R¹ togetherform a methylenedioxy group that is attached to two adjacent ring carbonatoms. In a further embodiment, n is 2 and R¹ is 6,7-diCH₃, 6,7-diOCH₃or 6,7-O—CH₂—O—. It is an embodiment that n is 2 and R¹ is 6,7-diCH₃ or6,7-diOCH₃.

In an embodiment, n is 3 and each R¹ is independently halo, C₁₋₄alkyl,C₂₋₄alkenyl or —X—C₁₋₄alkyl. In another embodiment, n is 3 and each R¹is independently CH₃ or OCH₃. In a further embodiment, n is 3 and R¹ is5,6,7-triOCH₃.

In an embodiment, the compound is selected from:

or a pharmaceutically acceptable salt, solvate and/or prodrug thereof.

In another embodiment, the compound is:

In a further embodiment, the compound is:

In an embodiment, A is O and the compounds of the disclosure areprepared, for example, by the reaction sequences shown in generalsynthetic scheme 1. A person skilled in the art could readily adapt sucha synthesis to prepare the corresponding compounds wherein A is S.

In an embodiment of the present disclosure, a compound of Formula I isprepared by a method comprising treating an acetanilide of Formula IIIwith DMF and POCl₃ under Vilsmeier Haack conditions to obtain a2-chloroquinoline 3-carboxaldehyde of Formula IV; performingClaisen-Schmidt condensation of the 2-chloroquinoline 3-carboxaldehydeof Formula IV with a substituted acetophenone of Formula V under basicconditions (for example, a catalytic amount of sodium methoxide or NaOH)to obtain a 3-(2-chloroquinolin-3-yl)-1-phenylprop-2-en-1-one of FormulaVI; and reacting the 3-(2-chloroquinolin-3-yl)-1-phenylprop-2-en-1-oneof Formula VI under conditions so that it undergoes O-nucleophilicsubstitution at the 2-chloro group (for example, treatment with aqueousglacial acetic acid under reflux) to provide the quinolone chalcone ofFormula I. The acetanilide of Formula III is optionally commerciallyavailable. Alternatively, the acetanilide of Formula III is preparedfrom the corresponding anilines according to standard procedures (Vogelet al., 1996). In the compounds of Formulae I to VI, R¹, R², R³ and nare as defined herein.

III. Compositions

The present disclosure also includes a composition comprising one ormore compounds of the present disclosure and a carrier. The compounds ofthe disclosure are optionally formulated into pharmaceuticalcompositions for administration to subjects or use in a biologicallycompatible form suitable for administration or use in vivo. Accordingly,the present disclosure further includes a pharmaceutical compositioncomprising one or more compounds of the present disclosure and apharmaceutically acceptable carrier.

The compounds of the disclosure can be administered to a subject or usedin a variety of forms depending on the selected route of administrationor use, as will be understood by those skilled in the art. In anembodiment, the one or more compounds of the disclosure are administeredto the subject, or used, by oral (including buccal) or parenteral(including intravenous, intraperitoneal, subcutaneous, intramuscular,transepithelial, nasal, intrapulmonary, intrathecal, rectal, topical,patch, pump and transdermal) administration or use and the compound(s)formulated accordingly. For example, the compounds of the disclosure areadministered or used in an injection, in a spray, in a tablet/caplet, ina powder, topically, in a gel, in drops, by a patch, by an implant, by aslow release pump or by any other suitable method of administration oruse, the selection of which can be made by a person skilled in the art.

In an embodiment, the one or more compounds of the present disclosureare orally administered or used, for example, with an inert diluent orwith an assimilable edible carrier, or enclosed in hard or soft shellgelatin capsules, or compressed into tablets, or incorporated directlywith the food of the diet. In an embodiment, for oral therapeuticadministration or use, the one or more compounds of the disclosure areincorporated with excipient and administered or used in the form ofingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. Oral dosage forms alsoinclude modified release, for example immediate release andtimed-release, formulations. Examples of modified-release formulationsinclude, for example, sustained-release (SR), extended-release (ER, XR,or XL), time-release or timed-release, controlled-release (CR), orcontinuous-release (CR or Contin), employed, for example, in the form ofa coated tablet, an osmotic delivery device, a coated capsule, amicroencapsulated microsphere, an agglomerated particle, e.g., asmolecular sieving type particles, or, a fine hollow permeable fiberbundle, or chopped hollow permeable fibers, agglomerated or held in afibrous packet. Timed-release compositions can be formulated, e.g.liposomes or those wherein the active compound is protected withdifferentially degradable coatings, such as by microencapsulation,multiple coatings, etc. Liposome delivery systems include, for example,small unilamellar vesicles, large unilamellar vesicles and multilamellarvesicles. In an embodiment, liposomes are formed from a variety ofphospholipids, such as cholesterol, stearylamine and/orphosphatidylcholines.

In another embodiment of the present disclosure, the one or morecompounds of the present disclosure are administered or usedparenterally. Solutions of the one or more compounds of the presentdisclosure are, for example, prepared in water optionally mixed with asurfactant such as hydroxypropylcellulose. In a further example,dispersions are prepared in glycerol, liquid polyethylene glycols, DMSOand mixtures thereof with or without alcohol, and in oils.Pharmaceutical forms suitable for injectable administration or useinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersions. A person skilled in the art would know how to preparesuitable formulations.

IV. Methods of Treatment and Uses

The compounds of the present disclosure are new therefore the presentdisclosure includes all uses for compounds of the present disclosure,including use in therapeutic methods, diagnostic assays, and as researchtools whether alone or in combination with another active pharmaceuticalingredient.

Together, the data from the present studies indicates that CTRcompounds, for example, CTR-20, are useful anticancer agents that can,for example, kill many different cancer cells includingcolchicine/paclitaxel-resistant, bortezomib-resistant, andmultidrug-resistant tumor cells with no notable ill-effects observed onnon-cancer cells and normal mouse organs.

Therefore, in an embodiment, the compounds of the present disclosure areuseful as medicaments. Accordingly, the present disclosure includes oneor more compounds of the present disclosure for use as a medicament.

The present disclosure also includes a method of treating cancercomprising administering one or more compounds of the disclosure to asubject in need thereof. The present disclosure also includes a use ofone or more compounds of the disclosure for treating cancer in asubject; a use of one or more compounds of the disclosure forpreparation of a medicament for treating cancer in a subject; and one ormore compounds of the disclosure for use to treat cancer in a subject.

In an embodiment, the cancer is breast cancer, leukemia, cervicalcancer, brain cancer, lung cancer, bladder cancer, kidney cancer,colorectal cancer, CNS cancer, melanomas, ovarian cancer, prostatecancer, multiple myeloma or other blood cancers. In another embodiment,the cancer comprises colchicine-resistant, paclitaxel-resistant,bortezomib-resistant, vinblastine-resistant and/or multidrug-resistanttumor cells.

Treatment methods or uses comprise administering to a subject or use ofan effective amount of one or more compounds of the disclosure,optionally consisting of a single administration or use, oralternatively comprising a series of administrations or uses. Forexample, the compounds of the disclosure are administered or used atleast once a week. However, in another embodiment, the compounds areadministered to the subject or used from one time per three weeks, orone time per week to once daily for a given treatment or use. In anotherembodiment, the compounds are administered or used 2, 3, 4, 5 or 6 timesdaily. The length of the treatment period or use depends on a variety offactors, such as the severity of the cancer, the age of the subject, theconcentration of the one or more compounds in a formulation, theactivity of the compounds of the present disclosure, and/or acombination thereof. It will also be appreciated that the effectiveamount of a compound used for the treatment or use may increase ordecrease over the course of a particular treatment regime or use.Changes in dosage may result and become apparent by standard diagnosticassays known in the art. In some instances, chronic administration oruse is required. For example, the one or more compounds of the presentdisclosure are administered or used in an amount and for durationsufficient to treat the subject.

The extent and/or undesirable clinical manifestations of cancer areoptionally lessened (palliated) and/or the time course of theprogression is slowed or lengthened, as compared to not treating thecancer.

The one or more compounds of the disclosure may be administered or usedalone or in combination with other therapeutic agents useful fortreating cancer; (optionally referred to herein as “anticancer agents”).When administered or used in combination with other known therapeuticagents, it is an embodiment that the one or more compounds of thedisclosure are administered or used contemporaneously with thosetherapeutic agents. As used herein the term “contemporaneous” inreference to administration of two substances to a subject or use meansproviding each of the two substances so that they are both biologicallyactive in the individual at the same time. The exact details of theadministration or use will depend on the pharmacokinetics of the twosubstances in the presence of each other, and can include administeringor using the two substances within a few hours of each other, or evenadministering or using one substance within 24 hours of administrationor use of the other, if the pharmacokinetics are suitable. Design ofsuitable dosing regimens is routine for one skilled in the art. Inparticular embodiments, two substances will be administered or usedsubstantially simultaneously, i.e., within minutes of each other, or ina single composition that contains both substances. It is a furtherembodiment that a combination of the two substances is administered to asubject or used in a non-contemporaneous fashion.

In an embodiment, the other agents are selected from the groupconsisting of mitotic inhibitors (for example, paclitaxel); bcl2inhibitors (for example, ABT-737); proteasome inhibitors (for example,bortezomib or calfilzomib); signal transduction inhibitors (for example,gefitinib, erlotinib, dasatinib, imatinib or sunitinib); inhibitors ofDNA repair (for example, iniparib, temozolomide or doxorubicin); andalkylating agents (for example, cyclophosphamide). In anotherembodiment, the other anticancer agent is paclitaxel.

The dosage of compounds of the disclosure can vary depending on manyfactors such as the pharmacodynamic properties of the compound, the modeof administration or use, the age, health and weight of the subject, thenature and extent of the symptoms of the cancer, the frequency of thetreatment or use and the type of concurrent treatment or use, if any,and the clearance rate of the compound in the subject. One of skill inthe art can determine the appropriate dosage based on the above factors.In an embodiment, the compounds of the disclosure are administered orused initially in a suitable dosage that is optionally adjusted asrequired, depending on the clinical response. As a representativeexample, oral dosages of one or more compounds of the disclosure willrange from less than 1 mg per day to 1000 mg per day for a human adultor an animal. In an embodiment of the present disclosure, thepharmaceutical compositions are formulated for oral administration oruse and the compounds are, for example in the form of tablets containing0.001, 0.01, 0.1, 0.25, 0.5, 0.75, 1.0, 5.0, 10.0, 20.0, 25.0, 30.0,40.0, 50.0, 60.0, 70.0, 75.0, 80.0, 90.0, 100.0, 150, 200, 250, 300,350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000mg of active ingredient per tablet. In an embodiment, the compounds ofthe disclosure are administered or used in a single daily dose or thetotal daily dose may be divided into two, three or four daily doses.

In the studies of the present disclosure, the combinational treatment ofMDR1 overexpressing KB-C-2 or MDA-MB231TaxR cells with paclitaxel andCTR-17, CTR-20, CTR-21 or CTR-32 showed synergistic effects and datafrom studies with engrafted mice showed that the combination of ½ dosesof CTR-20 and paclitaxel is more efficient than the full dose of eithercompound alone, without causing any notable ill-effects.

Accordingly, in embodiments wherein the one or more compounds of thepresent disclosure are administered or used in combination with one ormore other anticancer agents, the dosage of the one or more compounds ofthe present disclosure is optionally less than the dosage of the one ormore compounds of the present disclosure when administered or usedalone. In another embodiment, the dosage of the one or more compounds ofthe present disclosure is one half the dosage of the one or morecompounds of the present disclosure when administered or used alone.

In embodiments wherein the one or more compounds of the presentdisclosure are administered or used in combination with one or moreother anticancer agents, the dosage of the other anticancer agent isoptionally less than the dosage of the other anticancer agent whenadministered or used alone. In another embodiment, the dosage of theother anticancer agent is one half the dosage of the other anticanceragent when administered or used alone.

The following non-limiting examples are illustrative of the presentdisclosure:

EXAMPLES Example 1: Synthesis and Characterization of Compounds

I. Materials and Methods

The chemicals and solvents used were commercially available and were ofreagent grade. Melting points were determined in open glass capillarieson a Veego digital melting point apparatus and were uncorrected. Theinfrared (IR) spectra of the compounds were recorded on Schimadzu FT-IR8400S infrared spectrophotometer using an ATR accessory. ¹H NMR spectrawere recorded on a Bruker Avance II 400 spectrometer, using DMSO-d₆ assolvent and TMS as internal standard. Mass spectral analysis was carriedout using Applied Biosystem QTRAP 3200 MS/MS system in ESI mode.Reactions were monitored by TLC using pre-coated silica gel aluminumplates (Kieselgel 60, 254, E. Merck, Germany); zones were detectedvisually under ultraviolet irradiation.

II. General Synthetic Procedures

The synthesis of the quinolone chalcones of Formula I was carried outaccording to the reaction sequence illustrated in Scheme 2, wherein nwas 0, 1 or 2, R¹ was 6-OCH₃, 7-OCH₃, 8-OCH₃, 6,7-diOCH₃, 6-CH₃,6,7-diCH₃, 6-Cl, 6-Br or 7-Cl, R² was CH₃, C₂H₅ or CF₃ and R³ was absentor was OCH₃, OCF₃ or F, as appropriate for the compounds describedherein. The acetanilides (2a-2h) utilized in the synthetic route wereeither commercially available or synthesized from corresponding anilines(1a-1h) according to standard procedures (Vogel et al., 1996). Theacetanilides (2a-2h) were then treated with DMF and POCl₃ underVilsmeier Haack conditions to give 2-chloroquinoline 3-carboxaldehydes(3a-3h) (Meth-Cohn et al., 1981). Then, Claisen-Schmidt condensation ofthe 2-chloroquinoline-3-carbaldehydes (3a-3h) with the desiredsubstituted acetophenones (4a-4i) under basic conditions (a catalyticamount of sodium methoxide or NaOH) furnished3-(2-chloroquinolin-3-yl)-1-phenylprop-2-en-1-ones (QC-01-QC-25) in highyields (Dominguez et al., 2001; Li et al., 1995). The3-(2-chloroquinolin-3-yl)-1-phenylprop-2-en-1-ones upon treatment withaqueous glacial acetic acid under reflux conditions then underwent0-nucleophilic substitution at the 2-chloro group of the quinoline ringto give the corresponding quinolone chalcones of Formula I(CTR-17-CTR-40). The structural identity of the synthesized compoundswas established on the basis of their infrared (IR) spectroscopic, ¹HNMR and mass spectral data.

(a) General Procedure for the Synthesis of 2 chloro-3-formyl quinolines(3a-3h) (Meth-Cohn et al., 1981)

Acetanilide (2a)/substituted acetanilides (2b-2h) (0.05 mol) weredissolved in 9.6 ml of dimethyl formamide (0.125 mol) and to thissolution, 32 ml of phosphorus oxychloride (0.35 mol) was added graduallyat 0° C. The reaction mixture was taken in a round bottom flask (RBF)equipped with a reflux condenser fitted with a drying tube and washeated for 4-16 hours on oil bath at 75-80° C. The solution was thencooled to room temperature and subsequently poured onto 100 ml of icewater. The precipitate formed was collected by filtration andrecrystallized from ethyl acetate.

(b) General Procedure for the Synthesis of 2-chloroquinolinyl chalcones(QC-01-QC-25) (Dominguez et al., 2001; Li et al., 1995)

A mixture of 2-chloro-3-formyl quinolines (3a-3h) (1 mmol), therespective acetophenones (4a-4i) (1 mmol) and a base (sodium methoxide(catalytic) or sodium hydroxide (one pellet)) in methanol (4 ml) wasstirred at room temperature for 6-24 hr. The resulting precipitate wascollected by filtration, washed with water and recrystallized fromDMF-H₂O or EtOH-H₂O.

(c) General Procedure for the Synthesis of3-(3-oxo-3-phenylprop-1-enyl)quinolin-2(1H)-ones of Formula I (CTR-17 toCTR-40)

A suspension of the 3-(2-chloroquinolin-3-yl)-1-phenylprop-2-en-1-ones(QC-01-QC-25) (0.001 mol) in 70% acetic acid (10 ml) was heated underreflux for 4-6 hr. Upon completion of the reaction (as indicated by asingle spot in a TLC), the reaction mixture was cooled to ambienttemperature and the solid product precipitated out was filtered. Thefiltered product was washed with water, dried and recrystallized inmethanol or DMF/water.

III. Synthesis of Representative Compounds of the Disclosure

(a) Synthesis of(E)-3-(3-(2-Methoxyphenyl)-3-oxoprop-1-enyl)quinolin-2(1H)-one (CTR-17)2-Chloroquinoline-3-carbaldehyde (3a)

The title compound was prepared from acetanilide (2a) following theprotocol described above under subsection II(a) in the general procedurefor the synthesis of 2 chloro-3-formyl quinolines.

Yield 72%; M. P. 148-150° C. (Lit. 149° C.) (Srivastava and Singh,2005); FT-IR (ATR) υ (cm⁻¹): 3044 (Aromatic C—H), 2870 (aldehyde C—H),1684 (C═O), 1574 (C═N), 1045 (C—Cl); ¹H NMR (400 MHz, Chloroform-d): δ10.57 (s, 1H), 8.77 (s, 1H), 8.08 (d, J=8.5 Hz, 1H), 7.99 (d, J=8.1 Hz,1H), 7.90 (t, J=7.7 Hz, 1H), 7.66 (t, J=8.0 Hz, 1H); MS-API: [M+H]⁺ 192(calculated 191.01).

(E)-3-(2-Chloroquinolin-3-yl)-1-(2-methoxyphenyl)prop-2-en-1-one (QC-01)

The title compound was prepared by Claisen-Schmidt condensation of2-chloroquinoline-3-carbaldehyde (3a) with 2-methoxy acetophenone (4a)following the protocol described above under subsection II(b) in thegeneral procedure for the synthesis of 2-chloroquinolinyl chalcones.

Yield 62%; M.P. 113-115° C.; FT-IR (ATR) υ (cm⁻¹): 3067 (Aromatic C—H),1653 (C═O), 1603 (C═C), 1242 (C—O—C), 1045 (C—Cl); ¹H NMR (400 MHz,Chloroform-d): δ 8.43 (s, 1H), 8.08-8.00 (m, 2H), 7.87 (d, J=8.3 Hz,1H), 7.77 (t, J=7.8 Hz, 1H), 7.68 (dd, J=7.5, 1.8 Hz, 1H), 7.63-7.56 (m,1H), 7.55-7.45 (m, 2H), 7.11-7.01 (m, 2H), 3.94 (s, 3H); MS-API: [M+H]⁺324.1 (calculated 323.07).

(E)-3-(3-(2-Methoxyphenyl)-3-oxoprop-1-enyl)quinolin-2(1H)-one (CTR-17)

The title compound was prepared by refluxing3-(2-Chloroquinolin-3-yl)-1-(2-methoxyphenyl)prop-2-en-1-one (QC-01) inaqueous acetic acid (70%) following the protocol described above undersubsection II(c) in the general procedure for the synthesis of3-(3-oxo-3-phenylprop-1-enyl)quinolin-2(1H)-ones.

Yield 81%; M.P. 256-258° C.; FT-IR (KBr) υ (cm⁻¹): 3153 (NH), 1656(C═O), 1586, 1557 (C═C), 1240, 1020 (C—O—C); ¹H NMR (400 MHz, DMSO-d₆):(12.05 (s, 1H), 8.47 (s, 1H), 7.86 (d, J=16.0 Hz, 1H), 7.73 (d, J=7.9Hz, 1H), 7.60-7.44 (m, 4H), 7.34 (d, J=8.3 Hz, 1H), 7.26-7.19 (m, 2H),7.08 (td, J=7.4, 0.9 Hz, 1H), 3.87 (s, 3H); MS-API: [M+H]⁺ 306.1(calculated 305.1).

(b) Synthesis of(E)-3-(3-(2-Methoxyphenyl)-3-oxoprop-1-enyl)-6-methylquinolin-2(1H)-one(CTR-18) 2-Chloro-6-methylquinoline-3-carbaldehyde (3b)

The title compound was prepared from N-p-tolylacetamide (2b) followingthe protocol described above under subsection II(a) in the generalprocedure for the synthesis of 2 chloro-3-formyl quinolines.

Yield 75%; M.P. 122-123° C. (Lit. 123° C.) (Srivastava and Singh, 2005);FT-IR (ATR) υ (cm⁻¹): 3051 (Aromatic C—H), 2873 (aldehyde C—H), 1686(C═O), 1576 (C═N), 1055 (C—Cl); ¹H NMR (400 MHz, Chloroform-d): δ 10.55(s, 1H), 8.66 (s, 1H), 7.96 (d, J=8.5 Hz, 1H), 7.79-7.63 (m, 2H), 2.57(s, 3H); MS-API: [M+H]⁺ 206.02 (calculated 205.03).

(E)-3-(2-Chloro-6-methylquinolin-3-yl)-1-(2-methoxyphenyl)prop-2-en-1-one(QC-02)

The title compound was prepared by Claisen-Schmidt condensation of2-Chloro-6-methylquinoline-3-carbaldehyde (3b) with 2-methoxyacetophenone (4a) following the protocol described above undersubsection II(b) in the general procedure for the synthesis of2-chloroquinolinyl chalcones.

Yield 67%; M.P. 132-135° C.; FT-IR (ATR) υ (cm⁻¹): 3071 (Aromatic C—H),2833 (Aliphatic C—H), 1641 (C═O), 1597 (C═C), 1240, 1026 (C—O—C), 1047(C—Cl); ¹H NMR (400 MHz, Chloroform-d): δ 8.34 (s, 1H), 8.02 (d, J=15.9Hz, 1H), 7.91 (d, J=8.6 Hz, 1H), 7.68 (dd, J=7.6, 1.8 Hz, 1H), 7.65-7.55(m, 2H), 7.54-7.41 (m, 2H), 7.18-6.90 (m, 2H), 3.93 (s, 3H), 2.55 (s,3H); MS-API: [M+H]⁺ 338.1 (calculated 337.09).

(E)-3-(3-(2-Methoxyphenyl)-3-oxoprop-1-enyl)-6-methylquinolin-2(1H)-one(CTR-18)

The title compound was prepared by refluxing3-(2-Chloro-6-methylquinolin-3-yl)-1-(2-methoxyphenyl)prop-2-en-1-one(QC-02) in aqueous acetic acid (70%) following the protocol describedabove under subsection II(c) in the general procedure for the synthesisof 3-(3-oxo-3-phenylprop-1-enyl)quinolin-2(1H)-ones.

Yield 86%; M.P. 222-224° C.; FT-IR (KBr) υ (cm⁻¹): 3145 (NH), 1654(C═O), 1584, 1558 (C═C), 1241, 1019 (C—O—C); ¹H NMR (400 MHz, DMSO-d₆):δ 11.88 (s, 1H), 8.15 (s, 1H), 7.89 (d, J=15.9 Hz, 1H), 7.58 (d, J=15.9Hz, 1H), 7.50 (t, J=7.5 Hz, 2H), 7.44 (s, 1H), 7.32 (d, J=8.5 Hz, 1H),7.26 (d, J=8.4 Hz, 1H), 7.10 (d, J=8.3 Hz, 1H), 7.04 (t, J=7.4 Hz, 1H),3.90 (s, 3H), 2.39 (s, 3H), MS-API: [M+H]⁺ 320.1 (calculated 319.12).

(c) Synthesis of (E)-7-Methoxy-3-(3-(2-methoxyphenyl)-3-oxoprop-1-enyl)quinolin-2(1H)-one (CTR-19) 2-Chloro-7-methoxyquinoline-3-carbaldehyde(3c)

The title compound was prepared from N-(3-methoxyphenyl)acetamide (2c)following the protocol described above under subsection II(a) in thegeneral procedure for the synthesis of 2 chloro-3-formyl quinolines.

Yield 78%; M.P. 195-196° C. (Lit. 196° C.) (Srivastava and Singh, 2005);FT-IR (ATR) υ (cm⁻¹): 3053 (Aromatic C—H), 2879 (aldehyde C—H), 1688(C═O), 1583 (C═N), 1240, 1043 (C—O—C), 1051 (C—Cl); ¹H NMR (400 MHz,Chloroform-d): δ 10.51 (s, 1H), 8.66 (s, 1H), 7.85 (d, J=9.0 Hz, 1H),7.38 (s, 1H), 7.27 (dd, J=9.0, 2.5 Hz, 1H), 3.98 (s, 3H); MS-API: [M+H]⁺222.02 (calculated 221.02).

3-(2-Chloro-7-methoxyquinolin-3-yl)-1-(2-methoxyphenyl)prop-2-en-1-one(QC-03)

The title compound was prepared by Claisen-Schmidt condensation of2-Chloro-7-methoxyquinoline-3-carbaldehyde (3c) with 2-methoxyacetophenone (4a) following the protocol described above undersubsection II(b) in the general procedure for the synthesis of2-chloroquinolinyl chalcones.

Yield 63%; M.P. 178-180° C.; FT-IR (ATR) υ (cm⁻¹): 3073 (Aromatic C—H),2837 (Aliphatic C—H), 1666 (C═O), 1595 (C═C), 1227, 1020 (C—O—C), 1055(C—Cl); ¹H NMR (400 MHz, Chloroform-d): δ 8.35 (s, 1H), 8.02 (d, J=15.9Hz, 1H), 7.74 (d, J=9.0 Hz, 1H), 7.66 (dd, J=7.6, 1.9 Hz, 1H), 7.51(ddd, J=8.9, 7.5, 1.9 Hz, 1H), 7.43 (d, J=15.8 Hz, 1H), 7.34 (d, J=2.5Hz, 1H), 7.23 (dd, J=9.0, 2.5 Hz, 1H), 7.13-6.98 (m, 2H), 3.95 (s, 3H),3.93 (s, 3H); MS-API: [M+H]⁺ 354 (calculated 353.08).

(E)-7-Methoxy-3-(3-(2-methoxyphenyl)-3-oxoprop-1-enyl)quinolin-2(1H)-one (CTR-19)

The title compound was prepared by refluxing3-(2-Chloro-7-methoxyquinolin-3-yl)-1-(2-methoxyphenyl)prop-2-en-1-one(QC-03) in aqueous acetic acid (70%) following the protocol describedabove under subsection II(c) in the general procedure for the synthesisof 3-(3-oxo-3-phenylprop-1-enyl)quinolin-2(1H)-ones.

Yield 83%; M.P. 227-229° C.; FT-IR (KBr) υ (cm⁻¹): 3144 (NH), 1656(C═O), 1559 (C═C), 1167, 1021 (C—O—C); ¹H NMR (400 MHz, DMSO-d₆): δ11.96 (s, 1H), 8.40 (s, 1H), 7.85 (d, J=16.0 Hz, 1H), 7.60-7.42 (m, 3H),7.32-7.18 (m, 4H), 7.08 (t, J=7.4 Hz, 1H), 3.87 (s, 3H), 3.81 (s, 3H);MS-API: [M+H]⁺ 336.1 (calculated 335.12).

(d) Synthesis of (E)-6-Methoxy-3-(3-(2-methoxyphenyl)-3-oxoprop-1-enyl)quinolin-2(1H)-one (CTR-20) 2-Chloro-6-methoxyquinoline-3-carbaldehyde(3d)

The title compound was prepared from N-(4-methoxyphenyl)acetamide (2d)following the protocol described above under subsection II(a) in thegeneral procedure for the synthesis of 2 chloro-3-formyl quinolines.

Yield 63%; M. P. 145-146° C. (Lit. 146° C.) (Srivastava and Singh,2005); FT-IR (ATR) υ (cm⁻¹): 3053 (Aromatic C—H), 2829 (aldehyde C—H),1680 (C═O), 1574 (C═N), 1227, 1026 (C—O—C), 1051 (C—Cl); ¹H NMR (400MHz, Chloroform-d): δ 10.53 (s, 1H), 8.63 (s, 1H), 7.95 (d, J=9.2 Hz,1H), 7.50 (ddd, J=9.3, 2.9, 1.0 Hz, 1H), 7.18 (s, 1H), 3.94 (s, 3H);MS-API: [M+H]⁺ 222 (calculated 221.02).

3-(2-Chloro-6-methoxyquinolin-3-yl)-1-(2-methoxyphenyl)prop-2-en-1-one(QC-04)

The title compound was prepared by Claisen-Schmidt condensation2-Chloro-6-methoxyquinoline-3-carbaldehyde (3d) with 2-methoxyacetophenone (4a) following the protocol described above undersubsection II(b) in the general procedure for the synthesis of2-chloroquinolinyl chalcones.

Yield 69%; M.P. 226-228° C.; FT-IR (ATR) υ (cm⁻¹): 3071 (Aromatic C—H),2839 (Aliphatic C—H), 1666 (C═O), 1620 (C═C), 1234, 1020 (C—O—C), 1045(C—Cl); ¹H NMR (400 MHz, Chloroform-d): δ 8.32 (s, 1H), 8.00 (d, J=15.9Hz, 1H), 7.91 (d, J=9.3 Hz, 1H), 7.72-7.63 (m, 1H), 7.51 (ddd, J=8.9,7.4, 1.9 Hz, 1H), 7.48-7.36 (m, 2H), 7.18-6.99 (m, 3H), 3.95 (s, 3H),3.93 (s, 3H); MS-API: [M+H]⁺ 354.1 (calculated 353.08).

(E)-6-Methoxy-3-(3-(2-methoxyphenyl)-3-oxoprop-1-enyl)quinolin-2(1H)-one (CTR-20)

The title compound was prepared by refluxing3-(2-Chloro-6-methoxyquinolin-3-yl)-1-(2-methoxyphenyl)prop-2-en-1-one(QC-04) in aqueous acetic acid (70%) following the protocol describedabove under subsection II(c) in the general procedure for the synthesisof 3-(3-oxo-3-phenylprop-1-enyl)quinolin-2(1H)-ones.

Yield 83%; M.P. 227-229° C.; FT-IR (KBr) υ (cm⁻¹); 3155 (NH), 1652(C═O), 1597, 1558 (C═C), 1164, 1022 (C—O—C); ¹H NMR (400 MHz, DMSO-d₆):δ 11.91 (s, 1H), 8.37 (s, 1H), 7.78 (d, J=15.9 Hz, 1H), 7.64 (d, J=8.8Hz, 1H), 7.57-7.49 (m, 2H), 7.48-7.40 (m, 1H), 7.20 (d, J=8.5 Hz, 1H),7.07 (t, J=7.7 Hz, 1H), 6.89-6.81 (m, 2H), 3.85 (s, 3H), 3.84 (s, 3H);MS-API: [M+H]⁺ 336.1 (calculated 335.12).

(e) Synthesis of(E)-3-(3-(2,6-dimethoxyphenyl)-3-oxoprop-1-enyl)quinolin-2(1H)-one(CTR-25) 2-Chloroquinoline-3-carbaldehyde (3a)

The compound was prepared from acetanilide (2a) following the protocoldescribed above under subsection II(a) in the general procedure for thesynthesis of 2 chloro-3-formyl quinolines. Spectral data for the titlecompound is given above under subsection III(a).

(E)-3-(2-chloroquinolin-3-yl)-1-(2,6-dimethoxyphenyl)prop-2-en-1-one(QC-09)

The title compound was prepared by Claisen-Schmidt condensation of2-chloroquinoline-3-carbaldehyde (3a) with 2,6-dimethoxy acetophenone(4b) following the protocol described above under subsection II(b) inthe general procedure for the synthesis of 2-chloroquinolinyl chalcones.

Yield 71%; M.P. 199-201° C.; FT-IR (ATR) υ (cm⁻¹): 3004 (Aromatic C—H),2836 (Aliphatic C—H), 1650 (C═O), 1581 (C═C), 1223, 1020 (C—O—C), 1047(C—Cl); ¹H NMR (400 MHz, Chloroform-d): δ 8.42 (s, 1H), 7.98 (d, J=8.5Hz, 1H), 7.85 (d, J=8.3 Hz, 1H), 7.71-7.79 (m, 2H), 7.52-7.62 (m, 1H),7.35 (t, J=8.4 Hz, 1H), 7.00 (d, J=16.3 Hz, 1H), 6.63 (d, J=8.5 Hz, 2H),3.80 (s, 6H); MS-API: [M+H]⁺ 354.2 (calculated 353.08).

(E)-3-(3-(2,6-dimethoxyphenyl)-3-oxoprop-1-enyl)quinolin-2(1H)-one(CTR-25)

The title compound was prepared by refluxing3-(2-chloroquinolin-3-yl)-1-(2,6-dimethoxyphenyl)prop-2-en-1-one (QC-09)in aqueous acetic acid (70%) following the protocol described aboveunder subsection II(c) in the general procedure for the synthesis of3-(3-oxo-3-phenylprop-1-enyl)quinolin-2(1H)-ones.

Yield 65%; M.P. 236-238° C.; FT-IR (ATR) υ (cm⁻¹): 3149 (NH), 1667(C═O), 1591, 1558 (C═C), 1252, 1058 (C—O—C); ¹H NMR (400 MHz, DMSO-d₆):δ 12.00 (s, 1H), 8.41 (s, 1H), 7.66 (d, J=8.0 Hz, 1H), 7.47-7.55 (m,1H), 7.32-7.41 (m, 2H), 7.21-7.31 (m, 2H), 7.18 (t, J=7.6 Hz, 1H), 6.73(d, J=8.5 Hz, 2H), 3.69 (s, 6H); MS-API: [M+H]⁺ 336.2 (calculated335.12).

(f) Synthesis of(E)-3-(3-(2-ethoxyphenyl)-3-oxoprop-1-enyl)quinolin-2(1H)-one (CTR-32)2-Chloroquinoline-3-carbaldehyde (3a)

The title compound was prepared from acetanilide (2a) following theprotocol described above under subsection II(a) in the general procedurefor the synthesis of 2 chloro-3-formyl quinolines. Spectral data for thecompound is given above under subsection III(a).

(E)-3-(2-chloroquinolin-3-yl)-1-(2-ethoxyphenyl)prop-2-en-1-one (QC-16)

The title compound was prepared by Claisen-Schmidt condensation of2-chloroquinoline-3-carbaldehyde (3a) with 2-ethoxy acetophenone (4i)following the protocol described above under subsection II(b) in thegeneral procedure for the synthesis of 2-chloroquinolinyl chalcones.

Yield 73%; M.P. 128-130° C.; FT-IR (ATR) υ (cm⁻¹): 3055 (Aromatic C—H),2931 (Aliphatic C—H), 1669 (C═O), 1596 (C═C), 1238, 1037 (C—O—C), 1042(C—Cl); ¹H NMR (400 MHz, Chloroform-d): δ 8.42 (s, 1H), 7.98-8.07 (m,2H), 7.84 (d, J=8.0 Hz, 1H), 7.75 (t, J=7.6 Hz, 1H), 7.70 (d, J=7.5 Hz,1H), 7.55-7.61 (m, 2H), 7.48 (t, J=7.9 Hz, 1H), 7.05 (t, J=7.5 Hz, 1H),6.99 (d, J=8.3 Hz, 1H), 4.16 (q, J=7.0 Hz, 2H), 1.44 (t, J=7.0 Hz, 3H);MS-API: [M+H]⁺ 338.2 (calculated 337.09).

(E)-3-(3-(2-ethoxyphenyl)-3-oxoprop-1-enyl)quinolin-2(1H)-one (CTR-32)

The title compound was prepared by refluxing3-(2-chloroquinolin-3-yl)-1-(2-ethoxyphenyl)prop-2-en-1-one (QC-16) inaqueous acetic acid (70%) following the protocol described above undersubsection II(c) in the general procedure for the synthesis of3-(3-oxo-3-phenylprop-1-enyl)quinolin-2(1H)-ones.

Yield 77%; M.P. 199-201° C.; FT-IR (ATR) υ (cm⁻¹): 3128 (NH), 1651(C═O), 1597, 1555 (C═C), 1169, 1023 (C—O—C); ¹H NMR (400 MHz, DMSO-d₆):δ 12.02 (s, 1H), 8.39 (s, 1H), 8.00 (d, J=15.8 Hz, 1H), 7.68 (dd, J=8.0,1.3 Hz, 1H), 7.44-7.56 (m, 4H), 7.26-7.32 (m, 1H), 7.11-7.23 (m, 2H),7.02 (td, J=7.5, 1.0 Hz, 1H), 4.12 (q, J=7.0 Hz, 2H), 1.31 (t, J=6.9 Hz,3H). MS-API: [M+H]⁺ 320.2 (calculated 319.12).

(g) Synthesis of(E)-3-(3-(2-ethoxyphenyl)-3-oxoprop-1-enyl)-6-methoxyquinolin-2(1H)-one(CTR-40) 2-Chloro-6-methoxyquinoline-3-carbaldehyde (3d)

The title compound was prepared from N-(4-methoxyphenyl)acetamide (2d)following the protocol described above under subsection II(a) in thegeneral procedure for the synthesis of 2 chloro-3-formyl quinolines.Spectral data for the compound is given above under subsection III(d).

(E)-3-(2-chloro-6-methoxyquinolin-3-yl)-1-(2-ethoxyphenyl)prop-2-en-1-one(QC-24)

The title compound was prepared by Claisen-Schmidt condensation of2-chloro-6-methoxyquinoline-3-carbaldehyde (3d) with 2-ethoxyacetophenone (4i) following the protocol described above undersubsection II(b) in the general procedure for the synthesis of2-chloroquinolinyl chalcones.

Yield 74%; M.P. 151-153° C.; FT-IR (ATR) υ (cm⁻¹): 3058 (Aromatic C—H),2930 (Aliphatic C—H), 1655 (C═O), 1622 (C═C), 1233, 1021 (C—O—C), 1048(C—Cl); ¹H NMR (400 MHz, Chloroform-d): δ 8.31 (s, 1H), 7.99 (d, J=15.8Hz, 1H), 7.90 (d, J=9.3 Hz, 1H), 7.68 (d, J=7.8 Hz, 1H), 7.53 (d, J=15.8Hz, 1H), 7.47 (t, J=7.9 Hz, 1H), 7.39 (dd, J=9.3, 2.8 Hz, 1H), 7.01-7.09(m, 2H), 6.98 (d, J=8.3 Hz, 1H), 4.15 (q, J=6.9 Hz, 2H), 3.93 (s, 3H),1.42 (t, J=7.0 Hz, 3H); MS-API: [M+H]⁺ 368.2 (calculated 367.1).

(E)-3-(3-(2-ethoxyphenyl)-3-oxoprop-1-enyl)-6-methoxyquinolin-2(1H)-one(CTR-40)

The title compound was prepared by refluxing3-(2-chloro-6-methoxyquinolin-3-yl)-1-(2-ethoxyphenyl)prop-2-en-1-one(QC-24) in aqueous acetic acid (70%) following the protocol describedabove under subsection II(c) in the general procedure for the synthesisof 3-(3-oxo-3-phenylprop-1-enyl)quinolin-2(1H)-ones.

Yield 86%; M.P. 233-235° C.; FT-IR (KBr) υ (cm⁻¹): 3166 (NH), 1652(C═O), 1598, 1560 (C═C), 1245, 1023 (C—O—C); ¹H NMR (400 MHz, DMSO-d₆):δ 11.94 (s, 1H), 8.33 (s, 1H), 8.00 (d, J=15.8 Hz, 1H), 7.43-7.53 (m,3H), 7.17-7.26 (m, 3H), 7.15 (d, J=8.3 Hz, 1H), 7.02 (t, J=7.5 Hz, 1H),4.12 (q, J=6.8 Hz, 2H), 3.77 (s, 3H), 1.31 (t, J=7.0 Hz, 3H); MS-API:[M+H]⁺ 350.2 (calculated 349.13).

The foregoing syntheses are representative examples. Table 1 shows thechemical structures and names of 24 novel quinolone chalcone compoundswhich were synthesized and characterized in the present studies.

Example 2: In Vitro and In Vivo Studies of the CTR Compounds

I. Materials and Methods

Reagents

RPMI 1640, DME/F12, fetal bovine serum and antibiotic antimycoticsolutions (Pen/Strep/Fungiezone) were purchased from Hyclone (Logan,Utah). The antibodies specific for the following proteins were purchasedfrom Santa Cruz (Santa Cruz, Calif.): PARP (cleavage product), cdc2,phospho-cdc2 on Tyr15 or Thr161 residue, cyclin A, cyclin B, cyclin E,wee1, cdc25C, phospho-histone H3 (Ser10), α-tubulin, γ-tubulin andGAPDH. The antibodies specific for the following proteins were fromAbcam (Cambridge, UK): phospho-cdc25C on Thr48 or Ser216, securin,BubR1, and cdc20. Alexafluor 488 (anti-mouse) and 568 (anti-goat)conjugated IgG and DRAQ5/DAPI were purchased from MolecularProbes/Invitrogen. Tubulin polymerization kits (BK004P) and purifiedporcine tubulin (T240) were purchased from Cytoskeleton Inc. (Denver,Colo.). All reagents used for the experiments were of analytical grade.

Cell Lines and Cell Culture

All of the cell lines used were purchased from ATCC and cultured inRPMI-1640 supplemented with 10% fetal bovine serum and antibiotics (100units penicillin/100 μg/ml streptomycin), unless stated otherwise.1845B5 and MCF-10A non-cancer breast cell lines were cultured in DME/F12medium supplemented with 10% fetal bovine serum, antibiotics and growthfactors. KB-3-1 is a human epidermal carcinoma cell line and KB-C-2 isits isogenic multidrug-resistant cell line with over-expressingABCB1/P-gp. The KB-C-2 cell line was originally established in thepresence of increasing concentrations of colchicine. The human smallcell lung carcinoma H69 cell line and its multidrug-resistantMRP1-overexpressing isogenic H69AR cell line were purchased from ATCC.The H69AR cell line was established in the presence of increasingconcentrations of Adriamycin (doxorubicin). H69 cells grow as largemulti-cell aggregates, making it difficult to accurately count cellnumbers. Therefore, the cytotoxicity results obtained from H69AR cellswere compared to SW1271, a small cell lung carcinoma cell line without amultidrug-resistant phenotype. The IL-6 dependent bortezomib-resistantANBL6-BR cell line was further supplemented with 1 ng/ml of IL-6. TheMCF10AT1 and MCF10CA1a cell lines are isogenic to the MCF10A cell line,and obtained from Dr. Valerie Weaver at the Center for Bioengineeringand Tissue Regeneration, UCSF, CA, USA. MCF10AT1 is a premalignant cellline generated by transforming MCF10A with c-Ha-Ras; and MCF10CA1a wasisolated by selecting malignant cells after MCF10AT1 cells wereengrafted into mice (Liu & Lin 2004; Marella et al. 2009). The MCF10AT1and MCF10CA1a cells were cultured in DMEM supplemented with 10% FBS(volume/volume). MDA-MB231TaxR cell line was generated in house byculturing MDA-MB231 cells in gradually increasing doses of paclitaxelover one-year period, and finally maintained at 100 nM paclitaxel. Thedrug-resistant cells were cultured in the absence of drug for at leastone passage before carrying out experiments. All cells were maintainedin a humidified incubator at 37° C. (5% CO₂/95% air). Cell lineauthentication was performed using short tandem repeat (STR) profiling.

Sulforhodamine B (SRB)-Based Cytotoxicity Assay

For (anti)proliferation assays, 4,000-5,000 cells/well of the 96-wellclustered dish were incubated for 16 hours, as described previously (Huet al., 2008; Skehan et al., 1990). After 16 hours, culture medium wasreplaced with fresh medium containing different dilutions of testcompounds dissolved in DMSO. Some wells were treated with 100 μl of 10%trichloroacetic acid (TCA) as a negative control and sham (medium withdimethyl sulfoxide; DMSO) treated cells were used as a positive control.After 72 hours post-incubation, medium was removed and cells were fixedwith 10% TCA at 4° C. for 1 hour. TCA was removed and cells were washedwith cold tap water, and plate was air-dried, followed by addition of 50μl of 0.4% SRB staining solution to each well. After 30 minutesincubation, SRB staining solution was removed. Cells were washed with 1%acetic acid solution, and then washed with tap water to remove unboundstaining solution, followed by air-drying. 200 μl of 10 mM (pH10.5)trizma base buffer was added to each well to solubilise macromolecules.SRB stained macromolecules were determined at a 540 nm wavelength usingan automated plate reader (Synergy H4 Hybrid Multi-Mode MicroplateReader, BioTek, Winooski, Vt.). Cell growth (inhibition) was calculatedby the following formula:

% cells proliferation=[(AT−CT)/(ST−CT)]×100

wherein AT=absorbance of treated cells, CT=absorbance of negativecontrol cells, and ST=absorbance of sham treated cells. IC₅₀ values werecalculated from sigmoidal dose-response curves generated by twoindependent biological replicates, with quadruplicate in each set byusing Graph Pad Prism v.5.04 software. For combinational treatmentsagainst KB-C-2, MDA-MB231 or MB231TaxR cells, CTR compounds andpaclitaxel or ABT-737 were used at different concentrations which wereat or below the IC₅₀ values of single compounds. The combinational index(CI) was calculated as described previously (Chou 2006). If the CIvalues were less than, equal to or more than 1, it indicates asynergistic, additive or antagonistic effect respectively (Chou 2006).CI values were determined from four independent experiments.

Cell Cycle Analysis by Flow Cytometry

Approximately 1×10⁶ cells per plate were seeded and grown overnight.Cells were then treated the next morning with test compounds, andharvested at the scheduled post-treatment times. The cell pellet wascollected by centrifugation at 1,100 rpm (Allegra™ X-12 centrifuge,Beckman Coulter, Indianapolis, Ind.), followed by washing the cellstwice with PBS, and fixing them with 75% ethanol for 12-24 hours at −20°C. Ethanol was removed by centrifugation at 11,000 rpm (Allegra™ X-12centrifuge, Beckman Coulter); cells were suspended in PBS andcentrifuged again at 11,000 rpm in the same rotor. The PBS was thenremoved and the cell pellet was resuspended and stained for 1 hour withpropidium iodide (PI) staining solution (0.3% nonidet P-40, 100 μg/mlRNase A and 100 μg/ml PI in PBS). The DNA content in the differentphases of the cell cycle was analysed by flow cytometry using BeckmannCoulter Cytomics FC500 (Mississauga, ON, Canada). The reversibility ofdrug effects was determined as follow: HeLa cells treated with a CTRcompound for 12 hours were washed twice with 1×PBS, and then releasedthem into pre-warmed drug-free complete medium for scheduled durations.The cells were then examined by confocal microscopy for their morphologyor subjected to cell cycle analysis by flow cytometry after cells (DNA)were stained with propidium iodide.

Cell Synchronization

Synchronization at the G1/S border was achieved by double thymidineblock (DT). Briefly, exponentially growing cells were treated with 2.0mM thymidine for 18 hours, followed by incubation for 11 hours indrug-free complete medium, by which most cells are at mid-late G1 phase.The cells were then incubated for another 14 hours in 2.0 mM thymidineto arrest them at the G1/S border. To arrest cells at the prometa phase,cells were maintained for 18 hours in the complete medium containingnocodazole (50 ng/ml).

Immunofluorescent Staining

Cells on coverslips placed on the bottom of 35 mm tissue culture platesor 6-well clustered dishes were treated for 12-24 hours with the CTRcompounds to be tested. Subsequently, the cells were fixed with 100%methanol for 15 minutes and washed with 1×PBS three times. Cells werethen “blocked” with 3% BSA or 1% (v/v) FBS plus 1×PBST (1×PBS buffercontaining 0.1% (v/v) Triton X-100 or 0.2% Tween 20), and incubatedovernight at 4° C. with primary antibodies, with gentle agitation.Unbound primary antibodies were washed off with PBST, and a secondaryantibody was added for 1 hour in the dark. Secondary antibodies wereconjugated to Alexa 488 or 568. DNA was counterstained with DRAQ5 orDAPI. Subsequently, coverslips were washed three times with 1×PBST for10 minutes each, followed by mounting them onto slides with 90% glycerolin 1×PBS. Each slide was visualized with a Carl Zeiss 510 Meta laserscanning microscope or an Axioscope. Image analysis was done with an LSMimage examiner equipped with the microscopes (Carl Zeiss, Toronto, ON,Canada).

Western Blotting

Exponentially growing cells were collected by centrifugation at 1,100rpm (Allegra™ X-12 centrifuge, Beckman Coulter) at scheduled time pointspost-treatment. Cells were washed three times with PBS by centrifugationunder the same conditions, followed by cell lysis for 10-15 minutes onice in 100 μl Lysis buffer (150 mM NaCl, 5 mM EDTA, 1% triton X-100, 10mM tris pH 7.4, 1 mM PMSF, 5 mM EDTA and 5 mM protease inhibitor). Cellextracts were centrifuged at 11,000 rpm (Allegra™ X-12 centrifuge,Beckman Coulter) for 10 minutes at 4° C. Supernatant was collected andthe protein concentration was measured using a BCA assay kit accordingto the supplier's specifications (Thermo Fisher Scientific, Waltham,Mass.). Cell lysates were then diluted with 2× Iaemmli sample buffer andboiled for 5 minutes at 95-100° C. 30-40 μg protein was loaded on 8% or10% polyacrylamide gel and resolved by electrophoresis. Proteins werethen electronically transferred to a PVDF membrane for 75 minutes at 24volts, followed by “blocking” with 5% skim milk for 1 hour. Proteinswere incubated with primary antibody overnight at 4° C. in 0.1% TBSTbuffer containing 5% skim milk. The membrane was washed three times with0.1% TBST buffer and incubated for 1 hour with secondary antibody inTBST buffer containing 5% skim milk. The membrane was then washed withTBST buffer three times, and the signals were visualised on X-ray filmusing an ECL chemiluminescence kit (Super Signal West pico, ThermoFisher Scientific).

Immunoprecipitation

Cell lysates were prepared in 1×IP buffer (20 mM Tris-HCl, pH 7.5, 150mM NaCl, 1 mM EDTA and 1% (v/v) Triton X-100, supplemented with 10 mMsodium fluoride, 1 mM sodium orthovanadate and protease inhibitors) andpre-cleared for 3 hours at 4° C. by gentle agitation. Subsequently,immunoprecipitation was performed with an antibody overnight at 4° C.,followed by mixing with protein A/G agarose beads for an additional 5hours. The complex was washed five times with Lysis buffer, boiled for 5minutes, and resolved by SDS-PAGE, followed by immunostaining with anappropriate antibody.

Microtubule Polymerization Assay

The effects of the tested CTR compounds of the present disclosure on theassembly of purified tubulin were determined using a tubulinpolymerization kit according to the manufacturer's instructions(Cytoskeleton Inc., Denver, Colo.). Paclitaxel (provided in the samekit), nocodazole, and colchicine (Santa Cruz, Calif.) were used ascontrols for the assay. The absorbent-based assay kit is based on theprinciple that the light scattered by the microtubules is directlyproportional to the polymer mass of the microtubules when measured at37° C. at a wavelength of 340 nm. The fluorescence-based assay kit is onthe principle that fluorescent reporter molecules are incorporated intomicrotubules as the polymerization process being occurred. Thefluorescence enhancement was measured for one hour at one-minuteintervals, at the excitation of 350 nm and the emission of 430 nm.Absorbance or fluorescence was measured with an automated plate reader(Synergy H4 Hybrid Multi-Mode Microplate Reader, Bio-Tek).

Differential Tubulin Extraction

A two-step extraction procedure was used to separately isolate solubleand polymerised tubulin fractions from sham treated or treated withcompounds, as described previously (Tokesi et al., 2010). Briefly,exponentially growing cells were treated with 50 nM of paclitaxel, 50ng/ml nocodazole, 3.0 μM of CTR-17, or 1 μM of CTR-20 for 12 hours.Cells were then harvested and lysed with pre-warmed microtubulestabilizing buffer (80 mM PIPES, pH 6.8, 1 mM MgCl₂, 1 mM EGTA, 0.5%Triton X-100, 10% glycerol, and protease inhibitor cocktail). After abrief centrifugation at 2,500 rpm for 5 minutes at room temperature(Allegra™ X-12 centrifuge, Beckman Coulter), the tubulin heterodimers inthe soluble fractions were separated from supernatant by centrifugationas above. To ensure the soluble tubulin was completely extracted, thecell pellet was washed once again with the microtubule stabilizingbuffer; the supernatant fractions were pooled; and finally polymerizedtubulin complexes were extracted using microtubule destabilizing buffer(20 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 10 mM CaCl₂, andprotease inhibitor cocktail). The extract was cleared by centrifugationto obtain an insoluble microtubule fraction (2,500 rpm for 5 minutes atroom temperature with Allegra™ X-12 centrifuge, Beckman Coulter). Anequal amount of protein for each sample was resolved by SDS-PAGE,followed by Western blot and densitometry-based analyses usingAlphaEaseFC 4.0 software.

Determining the Dissociation Constant of CTR-Tubulin Binding

Purified tubulin (0.4 μM) was dissolved in 25 mM PIPES buffer (pH 6.8)and incubated in the presence or absence of different concentrations ofCTR-17 (or CTR-20) for 30 minutes at 37° C. The intrinsic fluorescenceof the tryptophan residues in the tubulin heterodimers was monitored byexcitation of the reaction mixture at 295 nm and the emission spectra atthe 315-370 nm wavelength range. All measurements were corrected for theinner filter using the formula F_(corrected)=F_(observed)×antilog[(A_(ex)+A_(em))/2], where A_(ex) and A_(em) are the absorbance of thereaction mixture at the excitation and emission wavelengths,respectively. Graph Pad Prism software was used to determine thedissociation constant of the CTR compounds binding to tubulin using thefollowing formula

${\Delta \; F} = \frac{\Delta \; {Fm}\; a\; x \times C}{{Kd} + C}$

wherein ΔF is the changes in fluorescence intensity of tubulin whenbound with the CTR compounds, ΔF_(max) is the maximum change in thefluorescence intensity when tubulin is bound with the compounds, C isthe concentration of the CTR compounds, and K_(d) is the dissociationconstant of the CTR compounds bound to tubulin.

Competitive Binding Assays

For the BODIPY FL vinblastine competition assay, 25 μM each of CTR-17,CTR-20, colchicine, and vinblastine was incubated with purified tubulinfor 1 hour at 37° C. Subsequently, BODIPY FL vinblastine was added tothe tubulin complex to a final concentration of 5.0 μM and the mixtureincubated for 30 minutes at 37° C. For the colchicine competition assay,tubulin was incubated with different concentrations of each compound for1 hour at 37° C. Subsequently, colchicine was added to the CTR-tubulinor vinblastine-tubulin equilibria to a final concentration of 1.0-8.0μM. Fluorescence was monitored using an automated plate reader (SynergyH4 Hybrid Multi-Mode Micro plate Reader, Bio-Tek). For the vinblastinecompetition assay, fluorescence was monitored by excitation of thereaction mixture at 490 nm and the emission spectra at the 510-550 nmrange. For the colchicine competition assay, the fluorescence of thetubulin complexes was determined with an excitation wavelength of 360 nmand emission wavelength at 430 nm. A modified Dixon plot was used toanalyze the competitive inhibition of colchicine binding to tubulin andto determine the inhibitory concentration (Ki) of the CTR compounds.

Molecular Modeling

The Molecular Operating Environment (MOE) (Chemical Computing Group Inc,Montreal, Quebec, Canada) was used to predict the interaction mode ofCTR compounds to the colchicine binding domain of the β-tubulin subunit.The crystal structure of the tubulin-colchicine complex (PDB Code: 1SA0) was used as the target structure and was subjected to energyminimization and protonation using the same software. The protocol fordocking was adopted from the MOE website, induced fit protocol was used.The best docking pose was determined based on the free energy forbinding. The contributions of H-bonds, hydrophobic, ionic and Van derWaals interactions were taken into consideration when calculating freeenergy values.

III. Animal Work Mice, Cells and Reagent/Protocols

Five-week old female CD-1 and ATH490 (strain code 490) athymic nude micewere purchased from Charles River (Quebec, Canada). The MDA-MB-231 humanmetastatic breast cancer cells were obtained from the American TissueCulture Collection (ATCC, Manassas, Va., USA). Cells were maintainedunder humidified conditions at 37° C. and 5% CO₂ in DMEM high glucosemedium (ATCC) supplemented with 10% fetal bovine serum and antibiotics.

For paclitaxel treatments, 40 mg/ml stock solution of paclitaxel (Sigma,MO) was prepared in DMSO. Just before administration to mice, thepaclitaxel stock solution was diluted ten-fold in a buffer containing10% DMSO, 12.5% Cremophor, 12.5% ethanol, and 65% saline-based diluent(0.9% sodium chloride, 5% polyethylene glycol, and 0.5% tween-80) whichis defined as vehicle (Huang et al., 2006). Alanine transaminase (ALT,SUP6001-c)/Aspartate transaminase (AST, SUP6002-c) color endpoint assaykits were purchased from ID Labs Biotechnology (London, Ontario,Canada). Elevation of ALT and AST levels in serum samples was used as anindicator of liver damage/injury.

Anti-Tumor Activity of CTR Compounds in Xenograft Mice

To determine the anti-tumor activity of CTR compounds in animals, axenograft model of human breast cancer cells in athymic nude mice wasestablished. Exponentially growing MDA-MB-231 metastatic breast cancercells were harvested and counted for inoculation into mice. Each mousewas subcutaneously injected at the flank with 10×10⁶ cells in 0.2 ml icecold 1×PBS. When tumor size reached 4-5 mm in diameter (n=4-5 pergroup), mice were randomly assigned into several groups as describedherein.

Animals were monitored for food and water consumption every day, andtheir body weights and tumor volumes were measured twice per week. Tumorvolumes were measured with a digital caliper and were determined byusing the following formula: ½ length×width². Blood samples werecollected via cardiac puncture and processed further for ALT and ASTmeasurements. The animals were then immediately euthanized by carbondioxide. Tumors and vital organs (spleen, kidney, liver and lung) werecollected and fixed in 10% buffered formalin at 4° C. overnight beforebeing processed for paraffin embedding. The paraffin-embedded blockswere then cut into 4-5 μm thick sections. Each section of tumors andorgans was stained with hematoxylin and eosin (H&E).

Toxicity Study in Animals

Changes in body weight, hemoglobin (Hb) and the amount and ratio ofalanine transaminase (ALT)/aspartate transaminase (AST) were used tomeasure toxic effects. In addition, vital organs (liver, spleen, kidneyand lung) were analyzed by fluorescent microscopy after they wereharvested, fixed, processed, paraffin-embedded, sectioned, and stainedas described above.

Statistical Analyses

All values are mean±S.E.M of at least three independent experiments.Analyses were performed using GraphPad Prism software (GraphPadSoftware, Inc). Comparison between the groups was made by p valuedetermination using one-way ANOVA. A p value of <0.05 was considered tobe statistically significant.

IV. Results

Table 2 contains a summary of the results from the initial screening offour CTR compounds using breast cancer cells (MDA-MB-231, MDA-MB-468,MCF-7) and non-cancer breast cells (184B5) determined by SRB assays. Ascan be seen from the results in Table 2, CTR-17, -18, -19, and -20 aremuch more effective than chloroquine or cisplatin, the two referencecompounds used in this experiment, CTR-17 and CTR-20 were observed topreferentially kill cancer cells over non-cancer cells up to 26 times(MDA-MB-468/K562 versus MCF-10A) and 24 times (HeLa versus MCF-10A),respectively. In contrast, cisplatin kills cancer and non-cancer cellswith similar efficacy.

Table 3 contains a summary of the results on the antiproliferationeffects of CTR-17 and CTR-20 on other cancer cell lines. All cell lineswere authenticated on Apr. 10 & Jul. 13, 2015 by STR profiling of gDNA.As can be seen from the results in Table 3, CTR-17 and CTR-20effectively kill many different cancer cells including brain cancer(U87MG), temozolomide-resistant glioblastoma (T98G), lung cancer(NCI-H1975, A549), multiple myeloma (RPMI-8229), urinary bladder cancer(UC3), and kidney cancer cell lines (HEK293T). The IC₅₀ values of CTR-20on the RPMI-8226-BR (bortezomib-resistant) and ANBL6-BR(bortezomib-resistant multiple myeloma) cell lines were also determinedin a separate experiment and found to be 0.28±0.03 μM and 0.76±0.28 μM,respectively.

Table 4 contains summary of the results on the anti-proliferationeffects of 16 novel CTR compounds (CTR-21 to CTR-40). These CTRcompounds killed MDA-MB231, MCF-7, HeLa and RPMI-8226 cancer cell lines,IC₅₀ values ranging from 5.34 nM (CTR-21 against RPMI-8226) to 2.69 μM(CTR-27 against MDA-MB231). For example, the IC₅₀ of CTR-21 in HeLa andRPMI-8226 cells was 11.93±1.40 and 5.34±0.89 nM, respectively.Similarly, CTR-32 was also effective as its IC₅₀ values were 12.88±0.35and 6.29±1.43 nM, respectively, against HeLa and RPMI-8226 cells.

As can be seen from the results in FIG. 1, CTR-20 induced cell death ina time- and dose-dependent manner when asynchronously growing HeLa S3cells were treated with CTR-20 at different concentrations (0, 0.5, 1.0,5.0 and 10.0 μM) for 24 or 72 hours (h). Cell survival/death wasdetermined by trypan blue exclusion assays. The treatment of HeLa cellswith 1 μM of CTR-20 resulted in ˜70% death by 72 hours post-treatment.

As can be seen from the results in FIG. 2, CTR-17 arrested cell cyclearound the G2/M phase. FIG. 2A shows flow cytometry profiles of HeLacells at 72 hours post-treatment with different concentrations (μM) ofCTR-17. FIG. 2B shows cell cycle profiles at different time points afterasynchronous HeLa cells were treated with 3.0 μM CTR-17. The majority ofHeLa cells arrested around the G2/M phase by 12 hours post-treatmentwith 3 μM CTR-17.

While not wishing to be limited by theory, the differential effects ofCTR-17 on cancer and non-cancer cells may be in part due to theirdifferences in cell cycle arrest in response to this compound. Twobreast cancer cell lines (MDA-MB-468 and MDA-MB-231) and one non-cancerbreast cell line (MCF-10A) were treated with 3.0 μM CTR-17 for 0-72hours, stained with propidium iodide, and their cell cycle profilesanalyzed by flow cytometry. The results are shown in FIG. 3. TheMDA-MB-468 metastatic breast cancer cells started to accumulate aroundG2/M by 6 hours post-treatment with 3 μM CTR-17, followed by massivecell death by 48 hours post-treatment. In MDA-MB-231, the G2/Mpopulation was accumulated much slower under the same conditions.Nevertheless, most of the MDA-MB-231 cells were arrested around G2/M by48 hours post-treatment. Although the G2/M population was enriched, thenon-cancer MCF-10A cells were never completely arrested in any cellcycle compartment.

As can be seen from the results in FIG. 4, CTR-20 selectively causedcell cycle arrest and cell death in cancer, but not in non-cancer cells.Asynchronous breast cancer cells (MDA-MB-231 and MCF-7) and theirmatching non-cancer breast cells (184B5) were treated with CTR-20 at 0.5or 1 μM for 72 hours. Cells were then collected, fixed and stained withpropidium iodide for cell cycle analysis by flow cytometry. Most of theMDA-MB-231 cells were dead within 72 hours in the presence of 1 μMCTR-20. While not wishing to be limited by theory, the profile of thesub-G1 DNA content suggests that the cell death may be by apoptosis.Most of the MCF-7 breast cancer cells were also arrested around G2/M,although they were not yet dead by 72-hours post-treatment. In contrastto the two cancer cell lines, 184B5 non-cancer breast cells were notsignificantly affected by 1 μM CTR-20 under the same experimentalconditions.

As can be seen from the results in FIG. 5, the treatment of cancer cellswith 1 μM CTR-20 resulted in cell cycle arrest around G2/M phase by 24hours and massive cell death by 72 hours post-treatment. Cells weretreated with 1 μM CTR-20 for MDA-MB-231 (FIG. 5A) and 0.5, 1.0 or 2.5 μMfor HeLa S3 (FIG. 5B) for 4, 8, 24, 48 and 72 hours (MDA-MB-231) or 24,48 and 72 hours (HeLa S3). At each time point, cells were collected,fixed with formaldehyde, and then stained with propidium iodide,followed by flow cytometry. As can be seen in FIG. 5, CTR-20 at 1 μMarrested both MDA-MB-231 and HeLa cells around G2/M by 24-hourpost-treatment. Most of these cells died with sub-G1 DNA content by72-hour post-treatment. K562 leukemic cells also showed a similarpattern of cell cycle arrest and cell death as we found it in a separateexperiment.

As shown in FIGS. 6 and 7, CTR-21 and CTR-32, similarly to CTR-17 andCTR-20, arrested cell cycle at G2/M. HeLa cells treated with 30 nM ofCTR-21 or CTR-32 transiently arrested at G2/M. However, cells neverreturned to G1 in a normal fashion when the concentrations of CTR-21 andCTR-32 were increase to 60 nM and 50 nM, respectively. In addition, theflow cytometry profiles at 48 and 72 hours post-treatment indicated that60 nM CTR-21 or 50 nM CTR-32 caused uneven cell division and cell death.

FIG. 8 shows that treatment of the MCF10A non-cancer cells with CTR-21(30 nM) or CTR-32 (50 nM) had no noticeable ill effects, except atransient arrest in G2/M at 12-hour post-treatment.

All four CTR compounds examined, (A) CTR-17, (B) CTR-20, (C) CTR-21 and(D) CTR-32, preferentially killed the fully malignant MCF10CA1a breastcancer cells over the premalignant MCF10AT1 and the non-cancer MCF10Abreast cells. For example, the cell survival rates at 0.39 μM ofCTR-17/CTR-20 were 39%/10%, 60%/20%, and 95%/75%, for MCF10Ala, MCF10AT1and MCF10A, respectively (FIGS. 9A & B). Similarly, the cell viabilityat 31.25 nM of CTR-21/CTR-32 was 10%/40%, 24%/70%, and 26%/87% forMCF10Ala, MCF10AT1 and MCF10A, respectively (FIGS. 9C & D).

CTR-17 caused monopolar centrosomes, defects in chromosome alignment,and uneven chromosomal segregation (FIG. 10). Cells were treated with3.0 μM CTR-17 for 12 hours, fixed in methanol, and stained with anantibody specific for γ-tubulin (green in a color image) or α-tubulin(red in a color image), and then the DNA counterstained with DAPI (bluein a color image). FIGS. 10A and 10B (higher magnification) showexemplary images of HeLa cells that were sham-treated or treated with 3μM CTR-17. White arrows on FIG. 10B denote uneven alignment/segregation.FIG. 10C shows exemplary images of HEK293T, MDA-MB-468 and MDA-MB-231cells that were treated with 3 μM CTR-17 in the same manner as in FIGS.10A and 10B. White arrows on FIG. 10C denote the failure of properalignment or uneven segregation of chromosomes. As can be seen from theresults shown in FIG. 10, most cells treated with 3.0 μM of CTR-17showed monopolar centrosomes, the failure of proper alignment at thecentre plate, and uneven chromosome segregation. These abnormalphenomena were observed in all of the cancer cell lines examined thusfar, including HeLa, HEK293T, MDA-MB-468 and MDA-MB-231 cell lines.

The cancer-specific increase of mitotic cells in response to CTR-17 wascorrelated with the accumulation of cells with monopolar centrosomes andabnormal chromosome alignment/segregation (FIG. 11, Table 5). Cells wereeither sham-treated or treated with 3.0 μM CTR-17 for 12 hours or 24hours, followed by analyses of cell cycle progression, centrosomeabnormalities, and chromosome alignment/segregation. FIG. 11 is a plotshowing that the treatment of cancer cells with CTR-17 resulted in theaccumulation of mitotic cells in a time-dependent manner. The mitoticindex was determined by fluorescence microscopic analysis of at least200 cells for each cell type, and data was expressed as percentagemean±S.E.M. of at least two independent experiments. Actual numberstaken from the plot in FIG. 11 are shown in Table 5. Table 5 does notinclude sham controls and non-cancer cells (MCF-10A and 184B5) sincetotal numbers of mitotic cells in these groups were too small to makemeaningful statistical comparison as these cells do not arrest atmitosis. As can be seen from this data, in response to 3 μM of CTR-17,cancer cells, but not non-cancer cells (MCF-10A, 184B5), wereaccumulated at mitotic phase with monopolar centrosomes or abnormalchromosome alignment/segregation.

Cells treated with CTR-20 showed monopolar centrosomes or abnormalchromosome alignment and segregation (FIG. 12). Asynchronously growingMCF-7, MDA-MB-231 and HeLa S3 cells were treated with 1 μM CTR-20 for 24hours. Cells were then collected, fixed with methanol, and incubatedwith an antibody specific for α-tubulin (green or red in color images),which was then counterstained DNA with DRAQ5 (red or blue in colorimages). The internal box in one of the MCF-7 samples in FIG. 12B showsuneven cell division. As can be seen from the exemplary images in FIG.12, cancer cells treated with 1 μM CTR-20 also showed multipolarcentrosomes and uneven cell division.

The number of cells containing monopolar centrosomes and chromosomeswith defective alignment and uneven segregation dramatically increasedin response to CTR-20 (FIG. 13, Table 6). As shown in FIG. 13, CTR-20caused cell cycle arrest at mitosis in a cancer cell-specific manner.Asynchronously growing cells were treated with 1 μM CTR-20 for 24 hours,followed by examining cells arrested in mitosis. The percentage ofmitotic cells was calculated based on the examination of at least250-400 cells. Cells treated with 1 μM CTR-20 for 24 hours on coverslipswere fixed with ice-cold methanol, immunostained with an α-tubulinantibody, and then counterstained DNA with DRAQ5 prior to observing byflorescent microscopy (Table 6). Table 6 does not include sham controlsand non-cancer cells (MCF-10A and 184B5) since the total numbers ofmitotic cells in these groups were too small to make meaningfulstatistical comparison as they do not arrest at mitosis. As can be seenfrom FIG. 13, CTR-20 at 1 μM caused the accumulation of mitotic cells incancer but not in non-cancer cells (MCF-10A, 184B5). Similarly, cellstreated with 1 μM CTR-20 accumulated monopolar centrosomes and defectivechromosomal alignment and segregation in a cancer-specific manner.

CTR-20 did not delay cells' entry into prometaphase/metaphase where theywere eventually arrested (FIG. 14). HeLa S3 cells growing on cover slipswere synchronized by double thymidine treatment (see materials andmethods). Cells were then released into fresh medium either in theabsence (Sham; FIG. 14A) or presence of 1.0 μM CTR-20 (FIG. 14B) for theduration of 7.5, 8.5, 9.5, 10, 10.5 or 11.5 hours. At the scheduled timepoints, cells were fixed with ice-cold methanol and immunostained withan antibody specific for α-tubulin, followed by counterstaining DNA withDRAQ5. Finally, cells were observed under a fluorescent microscope(Axio) at 40× objective. At least 15 fields were analyzed for eachsample. As can be seen from FIG. 14B, cells treated with 1 μM CTR-20normally progressed through the cell cycle until they reachedprometaphase; however, they were accumulated in prometaphase by 9.5-10hours and in metaphase by 11.5 hours post-G1/S in the presence of thedrug. Unlike the sham control (FIG. 14A), very little cells were at theanaphase-cytokinesis cell cycle compartment by even 11.5 hours post-G/Sin the presence of 1 μM CTR-20 (FIG. 14B), indicating that the metaphasearrest caused by CTR-20 is very effective. In contrast, sham-treatedcells (FIG. 14A) started to progress into anaphase/telophase andcytokinesis as early as 7.5 hours post-release from G1/S arrest. Mostcells in sham control (FIG. 14A) were in cytokinesis or interphase by11.5 hours. This data is consistent with the notion that cells arrestedin prometaphase in the presence of 1 μM CTR-20 eventually progressed tomitotic phase where they are “permanently” arrested.

Data from cells immunostained with antibodies specific for α-tubulin orγ-tubulin showed that both CTR-21 and CTR-32 caused defects in thechromosome alignment at metaphase (FIG. 15). Asynchronous HeLa cellswere treated with CTR-21 (30 nM) or CTR-32 (50 nM) for 12 hours,followed by staining DNA with Draq5 or immunestaining with antibodiesspecific for γ-tubulin or α-tubulin as shown on the top of the pictures.Chromosomes are not aligned properly at the center plate (white arrows).

FIG. 16 shows that HeLa cells arrested at mitosis in the presence ofCTR-21 or CTR-32 activated Bcl-X_(L) and apoptosis. Asynchronous HeLacells were treated with CTR-21 (15 or 30 nM) or CTR-32 (30 or 50 nM) for6, 12 or 24 hours (FIG. 16). The cell extracts of each sample weresubjected to protein separation by SDS-PAGE, followed by Westernblotting with antibodies specific for those listed at the right of gelspictures. High levels of cyclin B in the CTR treated samples as opposedto sham controls showed that the cells were arrested at M phase. Thestrong presence of high molecular weight Cdc25C (i.e., phosphorylated)at 12-hour post-treatment indicates that Cdk1/cyclin B was highly activeby that time; thus, the cells already entered M phase. The treatment ofcells with CTR-21 or CTR-32 caused the phosphorylation (i.e.,“activation”) of the Bcl-X_(L) anti-apoptotic protein by 6-hourpost-treatment. This was followed by cleavage of PARP proteins,suggesting that many cells underwent apoptosis by 24-hour post-treatmentwith CTR-21 (≥15 nM) or CTR-32 (≥30 nM).

Mitotic arrest caused by CTR-17 and CTR-20 was reversible. FIG. 17Ashows typical HeLa cell cycle histograms after they are treated withCTR-17 (3 μM; second from right) or CTR-20 (1 μM; far right) for 12hours (which is defined as time 0 post-release). At time 0 post-release,cells were washed twice with 1×PBS, followed by re-suspension of thecells in 10 ml of pre-warmed, drug-free medium for durations of 3, 6, 9or 12 hours (FIG. 17B). As can be seen from FIG. 17B, cells entered thecell cycle within 3 hours after CTR-17 and CTR-20 had been washed-off.This is in contrast to those cells still in culture medium containingCTR-17 and CTR-20 (FIGS. 11 and 13). Thus, this data shows that theeffects of CTR-17 and CTR-20 are reversible.

Like in the case of CTR-17 and CTR-20 (FIG. 17), the effect of CTR-21and CTR-32 is reversible (FIG. 18). HeLa cells entered to G1 of the nextcell cycle within 2 hours when those arrested at G2/M by the treatmentof CTR-21 (30 nM) or CTR-32 (50 nM) for 12 hours were washed PBS, andthen released into drug-free complete medium (FIG. 18A). Data fromconfocal microscopy (FIG. 18B) is consistent with the flow cytometrydata (FIG. 18A). Together, our data appear to suggest that the CTRcompounds may not have persistent side effects.

CTR-17 induces apoptosis in a cancer cell-specific manner (FIG. 19).Western blot analysis was carried out with an anti-PARP antibody at timepoints of 12, 24 or 48 hours post-treatment, using whole cell extractsprepared from asynchronous HeLa cells. As can be seen from FIG. 19,CTR-17 at 3.0 μM induced apoptosis by 48-hour post-treatment in HeLacells but not in 184B5 non-cancer cells. This data is consistent withthe data shown in Table 2.

CTR-17 caused neither impediment of DNA replication nor DNA damage. FIG.20A shows asynchronous HeLa cells treated with CTR-17 (3.0 μM) for 24hours, then fed with EdU (10.0 μM) for 1 hour immediately prior toharvesting them for analysis. The detection of EdU incorporated into DNAwas carried out by fluorescence microscopy. FIG. 20B shows cellimmunostaining with an antibody specific for γ-H2AX carried out todetect damaged DNA (i.e., damage repairing). Etoposide (50.0 μM) wasused as a positive control. As can be seen from the exemplary images inFIG. 20, under the experimental conditions used, EdU positive cells were25.4% and 22.0% for the sham control and CTR-17 (3.0 μM) treated cells,respectively. This data thus indicates that CTR-17 does not cause anyimpediment on DNA replication. This data is consistent with the datashown in FIG. 14. Data presented in FIG. 20B demonstrates that CTR-17does not cause any notable DNA damage.

Cells treated with CTR-17 arrested in mitosis, not in G2 (FIG. 21). AWestern blot analysis was carried out with whole cell extracts preparedfrom asynchronously growing HeLa cells. Equal amounts of proteins wereresolved by SDS-PAGE, and blotting was carried out with antibodiesspecific for those proteins listed at left of the gels shown in FIG. 21.Time points in hours (h) are post-treatment with 3.0 μM CTR-17. GAPDHwas used as a loading control. “p-” denotes phosphorylation. The Westernblot data shown in FIG. 21 demonstrated that CTR-17 causes cell cyclearrest in early M phase. This conclusion was derived from the fact that,judging from its phosphorylation on Tyr15, Cdk1 activity started toincrease around 12-hour post-treatment and was fully active until 48hours, the last time point examined. In an agreement with thisconclusion, Cdc25C activity culminated around the same time points,suggesting, while not wishing to be limited by theory, that theamplification of Cdk1 activation circuit was in high gear at least until24 hours post-treatment. However, Cdc25C was completely inactivated (seelevels of protein and phosphorylation on Thr48) by 48 hourspost-treatment, suggesting, while not wishing to be limited by theory,that Cdc25C is no longer needed to further amplify Cdk1 activity. Notethat the initial conclusion of cell cycle arrest around G2/M by CTR-17and CTR-20 was because data from flow cytometry was not detailed enoughto make a definite conclusion whether cells were in the G2 or M phase.

Cells did not exit mitosis in the presence of CTR-17. To accuratelyassess the effects of CTR-17 on cell cycle progression, HeLa cellssynchronised at the G1/S border by double thymidine (DT) block werereleased into cell cycle in the absence (sham) or presence of CTR-17(3.0 μM) for the duration in hours (h) indicated in FIG. 22. Cellstreated with CTR-17 progressed through the cell cycle in a similarfashion with the sham-treated control until 9 hours post-release fromthe G1/S arrest by double thymidine treatment. Unlike the sham control,however, the treated cells did not exit M phase. Instead, as can be seenin FIG. 22, most of cells treated with CTR-17 eventually died byapoptosis without entering into the G1 phase of the next cell cycle (48hours post-DT).

Data from the cell cycle study with synchronized cells demonstrated thatCTR-17 arrests cells in early mitosis. HeLa cells synchronized at theG1/S border by double thymidine (DT) block were released into completemedium at time 0 in the absence (sham; FIG. 23A) or presence (FIG. 23B)of 3.0 μM CTR-17 for the duration (hours) indicated. Equal amounts ofproteins were resolved by SDS-PAGE, followed by Western blotting withantibodies specific for proteins listed. “p-” denotes phosphoprotein.GAPDH was used as a loading control. Consistent with data fromasynchronous cells (e.g., FIG. 21), CTR-17 arrested cells in early Mphase when HeLa cells at the G1/S border were released into completemedium containing 3 μM CTR-17. Cdk1 and Cdc25C continue to be active inthe presence of CTR-17, which is manifested by the dephosphorylation ofCdk1 on Tyr15 and phosphorylation of Cdc25C on Thr48, at least up to 20hours post-release from double thymidine block. This coincided with thehigh levels of securin, cyclin B and histone H3 phosphorylation, whileonly negligible levels of cyclin E and cyclin A were observed. Inaddition, BubR1 was highly phosphorylated by 12 hours post-release.Together, while not wishing to be limited by theory, this data stronglysuggest that the cell cycle was arrested in the presence of CTR at thespindle checkpoint step (prior to APC-mediated securin degradation),presumably due to the failing of proper alignment of chromosomes at thecenter plate. This conclusion is supported by other data presented,including FIGS. 2, 3, 4, 5, 11, 13, 14, 21, 22, and 25.

Co-immunoprecipitation confirmed that CTR-17 causes cell cycle arrest atthe spindle checkpoint activation step. HeLa cells synchronised at theG1/S boundary by double thymidine (DT) block were untreated (sham),treated with 20 ng/ml nocodazole, or treated with 3.0 μM CTR-17 for theduration (h denotes hour(s)) indicated in FIG. 24. Total proteinextracts were subjected to immunoprecipitation with an anti-BubR1antibody, followed by protein separation by SDS-PAGE and Westernblotting with an anti-Cdc20 antibody to examine the interaction betweenBubR1 and Cdc20. Consistent with data from flow cytometry and Westernblotting, the data from co-immunoprecipitation (FIG. 24) demonstratedthat CTR-17 arrested cells at the spindle checkpoint step as BubR1 andCdc20 were associated in the presence of CTR-17. However, APC was notyet active. The cell cycle arrest point by CTR-17 is similar to that bynocodazole.

BubR1 accumulated at the kinetochore in the presence of CTR-17.Asynchronously growing HeLa cells were sham-treated or treated withCTR-17 (3.0 μM) for 12 hours, fixed, and then immunostained withantibodies specific for BubR1 or Cenp-B (centromere staining). As can beseen from FIG. 25, the accumulation of BubR1 at the kinetochoreindicates the lack of proper tension between the kinetochore and themitotic spindle/centrosome and, thus, perpetually extending the activityof spindle assembly checkpoint.

Both CTR-17 and CTR-20 inhibited tubulin polymerization. Purifiedporcine tubulin and 1.0 mM GTP were added to a reaction mixturecontaining 10.0 μM paclitaxel, 3.0 μM CTR-17, 1.0 μM CTR-20, or 5.0 μMnocodazole. Polymerization of tubulin was monitored every minute for onehour at 340 nm and 37° C. by spectrophotometry (FIG. 26). CTR-17 andCTR-20 caused an extended growth phase and took a long time to achievesteady state equilibrium in the microtubule polymerization reaction.This pattern is similar to that of nocodazole but different from that ofpaclitaxel, a microtubule stabilizing agent. While not wishing to belimited by theory, this data thus indicates that CTR-17 and CTR-20 areinhibitors of tubulin polymerization.

Both CTR-21 and CTR-32 are microtubule polymerization inhibitors. Datashown in FIG. 27 is from an in vitro microtubule assembly assay toexamine if CTR-21 and CTR-32 were microtubule inhibitors similarly toCTR-17 and CTR-20. Microtubule polymerization was monitored in relationto the incorporation of fluorescent reporter molecules intomicrotubules. The assay was carried out for one hour at 37° C., readingone minute intervals by spectrophotometry. CTR-21 and CTR-32 were usedat two different concentrations, 100 nM and 1.0 μM. At 10 nM, both ofthem inhibited microtubule polymerization to a similar degree of CTR-20at 1.0 μM, indicating that CTR-21 and CTR-32 are stronger microtubuleinhibitors than CTR-20. Among the two, CTR-21 appears to be moreeffective than CTR-32 in inhibiting microtubule polymerization.

CTR-17 and CTR-20 decreased the polymerized pool of tubulin (FIG. 28).HeLa, MDA-MB-231 and MDA-MB-468 cells were sham-treated, treated with50.0 nM paclitaxel (Tax), 50.0 ng/ml nocodazole (Noc), 3.0 μM CTR-17, or1.0 μM CTR-20 for 12 hours. Cell lysates were separated intopolymerization (Pol) and soluble (Sol) fractions, and equal amounts ofproteins were resolved by SDS-PAGE, followed by immunoblotting with anantibody specific for α-tubulin. Bands (upper panel of FIG. 28A) werequantified with densitometry and expressed in a graph form (lower panelof FIG. 28A). The sum of soluble and polymerized fractions is 1.0. HeLacells treated with different concentrations of CTR-17 or CTR-20 weresubjected to fractionation and immunoblotting as described for FIG. 28A.Both CTR-17 and CTR-20 reduced the tubulin polymer fraction similarly tonocodazole. This fractionation pattern is in a stark contrast withpaclitaxel, which increases the tubulin polymer fraction. The reductionof tubulin polymerization by CTR-17 was dose-dependent within the rangeof concentrations of 3-6 μM (FIG. 28B). However, the effect of CTR-20was already saturated at the 1.0 μM concentration, indicating thatCTR-20 is a stronger inhibitor of tubulin polymerization (FIG. 28B).

Both CTR-17 and CTR-20 bound to tubulin. As can be seen from FIGS. 29Aand 29B, respectively, CTR-17 and CTR-20 quenched the intrinsictryptophan fluorescence of tubulin in a dose-dependent manner. Purifiedtubulin dissolved in 25 mM PIPES buffer was incubated in the presence orabsence of different concentrations of CTR-17 or CTR-20 for 30 minutesat 37° C. Fluorescence was monitored by excitation of the reactionmixture at 295 nm, and the emission spectra were recorded from 315 to370 nm. FIGS. 29C and 29D, respectively, show the change in fluorescenceintensity plotted against the drug concentrations of CTR-17 and CTR-20to determine the dissociation constant. ΔF (y-axis) is the change influorescence intensity of tubulin when bound by the CTR compounds. Dataare an average of five independent experiments. Both CTR-17 and CTR-20bound to tubulin, in a dose-dependent manner.

Both CTR-17 and CTR-20 inhibited the binding of colchicine to tubulin(FIG. 30). The CTR compounds tested, similar to colchicine, did not bindto the vinblastine binding site on the tubulin. 25 μM each ofcolchicine, CTR-17, CTR-20, or vinblastine was incubated with tubulinfor 1 hour to promote the formation of complexes between tubulin andeach of these compounds. The resultant complexes were incubated for 30minutes with 5 μM of the fluorescent BODIPY FL-vinblastine to determineif the binding of each compound to tubulin was in competition withvinblastine. CTR-17 binds to tubulin at or near the colchicine-bindingsite. The tubulin-fluorescent colchicine complex was incubated withincreasing concentrations of either vinblastine or CTR-17. CTR-17 butnot vinblastine competed with (fluorescent) colchicine. The CTRcompounds depressed the fluorescence of the colchicine-tubulin complexin a dose-dependent manner. Tubulin was incubated with differentconcentrations of CTR-17 or CTR-20 for 1 hour, in three separate setswith different concentrations of colchicine as indicated in FIG. 30.Inhibitory constants of CTR-17 and CTR-20 were determined. Thefluorescence intensity of the final tubulin complex (FIGS. 30C and 30D)was used to determine the inhibitory concentration (Ki) utilizing amodified Dixon plot (FIGS. 30E and 30F). In FIG. 30, F is thefluorescence of the complexes of CTR-17 (or CTR-20)-colchicine-tubulinor vinblastine-colchicine-tubulin complex, and F0 is the fluorescence ofthe colchicine-tubulin complex. Data are average of at least fourindependent experiments. The data of FIG. 30, while not wishing to belimited by theory, suggests that the binding sites of both CTR-17 andCTR-20 on the tubulin may overlap with that of colchicine but not thatof vinblastine.

FIG. 31A shows the result of molecular docking predicting that thetubulin-binding sites of colchicine (blue in color image; medium grey inFIG. 31A), CTR-17 (green in color image; lighter grey in FIG. 31A),CTR-20 (magenta in color image; darker grey in FIG. 31A) andpodophyllotoxin (yellow in color image; light grey in FIG. 31A) werevery close, but not with that of vinblastine (red in color image;darkest grey in FIG. 31A). The 3D X-ray structure of tubulin (PDB code:1 SA0) was used in this study. FIG. 31B shows the chemical structures ofcolchicine (blue in color image; darkest grey in FIG. 30B), CTR-17(green in color image; light grey in FIG. 31B), CTR-20 (magenta in colorimage; dark grey in FIG. 31B) and podophyllotoxin (light red in colorimage; lightest grey in FIG. 31B) are shown to aid the visualization ofthe close overlap when bound to their respective binding sites on thetubulin. In sum, while not wishing to be limited by theory, data frommolecular modeling showed that the tubulin sites bound by CTR-17 andCTR-20 essentially overlap with those of colchicine and podophyllotoxin,but are completely different from that of vinblastine.

The predicted interaction between the tubulin heterodimer (PDB code:1SA0) and colchicine (A), CTR-20 (B), and CTR-17 (C) is shown in a 3Dpattern in FIG. 32. 2D ligand interaction diagrams in FIG. 32 showpotential chemical interactions between amino acids and compounds withina distance of 4 Å to colchicine (A′), CTR-20 (B′), or CTR-17 (C′). Thereare three H-bonds between tubulin and colchicine, while one and twoH-bonds between tubulin-CTR-17 and tubulin-CTR-20, respectively. Thedirection of arrows shows the electron donor in hydrogen bonding.H-bonds are formed through a side chain and an amino acid backbone,respectively. A number of hydrophobic and polar residues overlap betweencolchicine and the CTR compounds in binding to tubulin. The color codesare: dark grey (red in a color image) square boxes are tubulin aminoacids that are common in binding to colchicine and CTR-20; lightest grey(yellow in a color image) boxes are those common in binding tocolchicine and CTR-17; and light grey (blue in a color image) boxes arethose common in binding to CTR-17 and CTR-20. Non-covalent and Van derWaals interactions stabilize the binding between tubulin and thesecompounds. In sum, data obtained from molecular modeling showed that thebinding mode of colchicine, CTR-17 and CTR-20 to tubulin is very similaras they often bind to the same amino acid residues on tubulin. However,they also show differences in the binding mode. For example, colchicineforms three H-bonds, and CTR-17 and CTR-20 form only one and twoH-bonds, respectively. While not wishing to be limited by theory, it ispossible that these differences are directly relevant, for example, toefficacy, toxicity and reversibility of the compounds.

CTR-17 and CTR-20 are effective against multidrug-resistant cancercells. Western blotting of whole cell extracts prepared from theparental KB-3-1 and MDR1-overexpressing KB-C-2 isogenic cell lines areshown in FIG. 33A. Western blotting of whole cell extracts prepared fromthe parental H69 and MRP1-overexpressing H69AR isogenic cell lines areshown in FIG. 33B. The data in Table 7 show that CTR-17 and CTR-20 killparental (KB-3-1) and MDR1-overexpressing multidrug-resistant cells(KB-C-2) with a similar potency, and both of these CTR compoundspreferentially kill MRP1-overexpressing multidrug-resistant cells(H69AR) over matching small cell lung cancer cells (SW-1271). An SRBassay was used to determine the anti-proliferation effects. Colchicine,paclitaxel and vinblastine are largely ineffective in killingmultidrug-resistant cells. Data shown in FIG. 33 and Table 7 demonstratethat both CTR-17 and CTR-20 kill KB-3-1 (cervical carcinoma) and itsMDR1-overexpressing multidrug-resistant isogenic cells (KB-C-2) withsimilar potency, while colchicine, paclitaxel, and vinblastine kill thedrug-resistant cells at least 10 fold less effectively thannon-resistant cells. Furthermore, both of the CTR compoundspreferentially killed the MRP1-overexpressing, multidrug-resistant H69ARcells over a matching non-cancer small cell lung cancer cell line(SW-1271).

As can be seen from FIG. 34, both CTR-17 and CTR-20 show synergisticeffects when combined with paclitaxel. The KB-C-2 multidrug-resistantcells were subjected to an antiproliferation study with combinations ofdifferent doses of CTR compounds and paclitaxel. Data from SRB assayswas used to construct a sigmoidal dose-response curve, from which themedian effect dose (Dm), fraction affected (Fa) and slope of the curve(m) were determined. These values were then used to determine thecombination effect between CTR-17/-20 with paclitaxel, as outlined inthe methodology section. Part of the CTR-17 data presented in Table 8 isshown in a graph form in FIG. 34A. Lanes denote: 0.65 μM CTR-17 (lanes 1& 4), 23 nM paclitaxel (lane 2), 0.65 μM CTR-17 plus 23 nM paclitaxel(lane 3), 5.75 nM paclitaxel (lane 5), and 0.65 μM CTR-17 plus 5.75 nMpaclitaxel. Part of CTR-20 data presented in Table 8 is shown in a graphform in FIG. 34B. Lanes denote: 0.25 μM of CTR-20 (lanes 1 & 4), 23 nMpaclitaxel (lane 2), 0.25 μM CTR-20 plus 23 nM paclitaxel (lane 3), 11.5nM paclitaxel (lane 5), and 0.25 μM CTR-20 plus 11.5 nM paclitaxel (lane6). CI denotes combination index. CI<1.0, CI=1.0 and CI>1.0 aresynergistic, additive and antagonistic, respectively (Chou, 2006). Formore detail, see Table 8. Data presented are mean±S.E.M value oftriplicates of at least four independent experiments. As seen from thedata in FIG. 34 and Table 8, both CTR-17 and CTR-20 showed synergisticcell killing effects when used in combination with paclitaxel on KB-C-2multidrug-resistant cells. Note that the combination index (CI) of 0.65μM CTR-17 and 23.0 nM paclitaxel was 0.71±0.08, and that of 0.25 μMCTR-20 and 11.5-23.0 nM of paclitaxel was 0.69. Thus, the combination ofCTR compounds and paclitaxel can be substantially synergistic on theMDR1-overexpressing multidrug-resistant KB-C-2 (and, while not wishingto be limited by theory, other) cells.

Data in FIG. 35 shows that the MDR1-overexpressing paclitaxel-resistantMDA-MB231TaxR is sensitive to CTR-17, CTR-20, CTR-21 and CTR-32. Thepaclitaxel-resistant MDA-MB231TaxR cell line was generated in house byculturing the triple-negative MDA-MB231 metastatic breast cancer cellline in the incrementally increased concentrations of paclitaxel overone-year period, until the cells grow and proliferate in the mediumcontaining 100 nM of paclitaxel. Subsequently, the cells were dosed with100 nM paclitaxel once every month and removed from the drug at leastfor one passage before being used for an experiment. FIG. 35A shows theWestern blotting of MDA-MB231TaxR cells at different levels ofpaclitaxel resistance alongside the MDA-MB231 parental cells (WT) forthe expression of P-glycoprotein (MDR1): 2.0, 10.0, 15.0, 30.0 and 100.0nM are cells selected at the concentrations of paclitaxel at 2.0, 10.0,15.0, 30.0 and 100.0 μM, respectively. Parental MDA-MB231 cells (WT) donot express P-glycoprotein; however, the level of P-glycoproteinexpression increases with increasing levels of resistance in the TaxRcells (FIG. 35A). Data in FIG. 35B shows that colchicine, CTR-17,CTR-20, CTR-21, and CTR-32 kill MDA-MB231TaxR cells (selected in 100.0nM paclitaxel) to the same degree as the parental MDA-MB231 cells.However, MDA-MB231TaxR cells were resistant to paclitaxel andvinblastine by approximately 114 and 15 folds, respectively, suggestingthat MDA-MB231TaxR cells are multidrug resistance in nature.

Data in FIG. 36 shows that the bortezomib-resistant RPMI-8226BTZRmultiple myeloma cells are sensitive to CTR-20, CTR-21 and CTR-32. Thebortezomib-resistant RPMI-8226BTZR cell line was developed in house byculturing the RPMI-8226 multiple myeloma cell line in the graduallyincreased concentrations of bortezomib, a proteasome inhibitor targetingβ5 peptide of the 20S catalytic subunit. The RPMI-8226BTZRover-expresses β1, β2 and β5 peptides of the 20S subunit. FIG. 36 showsthat RPMI-8226BTZR is approximately 28-fold more resistant tobortezomib, compared to the RPMI-8226 parental cells. However,RPMI-8226BTZR is sensitive to CTR-20, CTR-21 and CTR-32 (FIG. 36).

When combined with paclitaxel, CTR-20, CTR-21 and CTR-32 showed synergyin killing the paclitaxel/multidrug-resistant MDA-MB231TaxR cells (FIG.37 and Table 9). It was found previously that both CTR-17 and CTR-20 aresynergistic with paclitaxel in killing the MDR1-overexpressing KB-C-2cells (FIG. 34 and Table 8). Data in FIG. 37 and Table 9 show thecombinational effects of paclitaxel and CTR-20 (A), CTR-21 (B) andCTR-32 (C) against MDA-MB231TaxR cells. For this set of experiments, wecombined 4-5 different doses of paclitaxel with a single dose of CTR-20,CTR-21 or CTR-32. Cell viability was always greater than 50% when eachof these drugs was used singly at the doses used. However, the cellviability reduced at the same doses when combined with paclitaxel andCTR-20, CTR-21 or CTR-32. For example, the combination of 0.3 μM ofCTR-20 and 37.5 nM paclitaxel killed MDA-MB231TaxR cells approximately56% (Table 9). The combination of 23.4 nM of CTR-21 and 18.75 nMpaclitaxel killed MDA-MB231Tax cells 58% (Table 9). Finally, thecombination of 23.4 nM of CTR-32 and 18.75 nM paclitaxel killed 52% ofMDA-MB231TaxR cells (Table 9). These data are translated intocombinational index (CI) of 0.59, 0.35 and 0.27, respectively (Table 9and FIG. 37), showing that these combinations are synergistic. Sincepaclitaxel is generally toxic, the synergistic effects of paclitaxel-CTRcombinations at low drug concentrations against multidrug-resistantcancer cells will provide new opportunities of controllingdrug-resistant cancer with low side effects.

Data in FIG. 38 and Table 10 show that the combination of CTR-20 andABT-737 is synergistic against MDA-MB231 triple-negative metastaticbreast cancer cells. We examined the combinational effects CTR-20 andthe inhibitor of anti-apoptotic Bcl2 family proteins. Data from all ofthe different combinations by three different doses of CTR-20 and twodifferent doses of ABT-737 showed synergistic effects on MDA-MB231cells. In particular, the combination of 0.2 μM of CTR-20 and 6.25 μM ofABT-737 showed the CI value of 0.07, a high degree of synergism.Similarly, the CI of the 0.4 μM of CTR-20 and 6.25 μM of ABT-737combination was 0.10. At these combinations, MDA-MB231 cell populationwas eliminated completely (Table 10 and FIG. 38).

Data in FIG. 39 shows that the combination of 0.4 μM CTR-20 (CTR) and6.25 μM ABT-737 (ABT) completely kill off the MDA-MB231 population by 72hours post-treatment. As a single regimen, ABT-737 up to 6.25 μM did notalter cell cycle progression in any substantial way. However, MDA-MB231cells were “permanently” arrested at G2/M in the presence of 0.4 μMCTR-20 plus 6.25 μM ABT-737, leading to apoptotic cell death (manifestedby the presence of sub-G1 DNA contend) by 72 hours after thecombinational treatment.

The combination of CTR-20 and ABT-737 induces apoptosis through cellcycle arrest at M and suppressing anti-apoptotic Bcl-X_(L) and Mcl-1.Data in FIG. 40 shows: (1) that the levels of cyclin B and Bcl-X_(L)phosphorylation on the serine 62 residue substantially increases in thepresence of 0.4 μM of CTR-20, indicating that the cell cycle arrested atG2/M and anti-apoptotic pathway was suppressed (black arrows); (2) thatthe combination of 0.2-0.4 μM CTR-20 and 6.25 μM ABT-737 furthersuppressed the anti-apoptotic pathway by downregulating Mcl-1 (whitearrows); and (3) the combination of CTR-20 (0.2-0.4 μM) and ABT-737(3.13-6.25 μM) effectively induced apoptosis as manifested by thecleavage of PARP and caspase 3.

Data in FIG. 41 shows that CTR-20 effectively killed or inhibited cellproliferation of all the cell lines included in the NCI 60 cancer panel.Data from an SRB-mediated cell survival assay carried out by the USnational Cancer Institute show that cells treated with 10 μM CTR-20 for48 hours effectively killed/inhibited proliferation of all the 60 cancercell lines including in the NCI-60 panel: six leukemia cell lines(ranging from −2.46 to +10.88%), nine non-small cell lung cancer celllines (−11.97 to 38.93%), seven colorectal cancer cell lines (+4.37 to+23.11), six CNS cancer (−+11.48 to +17.04%), nine melanomas (−10.05 to+50.45%), seven ovarian cancer cell lines (+1.18 to +50.80%), sevenrenal cancer cell lines (−7.90 to +42.01%), two prostate cancer celllines (+10.76 to +22.06), and six breast cancer cell lines (−2.02 to+18.91%).

As can be seen from FIG. 42, both CTR-17 and CTR-20 showed effectiveanti-tumor activity in a xenograft model. FIG. 42A shows changes intumor size (volume in mm³) in response to drug treatments, alone or incombination with paclitaxel. Experimental protocol and data are shown inTables 11 and 12, respectively. The antitumor activities of CTR-18 andCTR-19 are shown in Table 13. Values are means±S.E.M. “D” denotes day(s)post-treatment. FIG. 42B shows exemplary images of representative ATH490athymic mice engrafted with MDA-MB-231 human metastatic breast cancercells that were vehicle only or treated with drugs as indicated. Numbersin brackets are mg/kg body weight. As can be seen from FIG. 42 and theTables 11 and 12, both CTR-17 and CTR-20 showed strong antitumoractivity against MDA-MB-231 metastatic breast cancer in the mousexenograft model. The combination of a ½ dose of CTR-17 (or CTR-20) witha ½ dose of paclitaxel was considerably more effective than full dose ofCTR-17, CTR-20, or paclitaxel alone. Although all four CTR compounds(CTR-17, -18, -19 and -20) showed antitumor activities, CTR-20 shows thegreatest antitumor activity with mice engrafted with MDA-MB-231metastatic breast cancer cells. This result is consistent with that ofthe in vitro study.

Data from body weight analysis indicates that CTR compounds are nottoxic to mice (FIG. 43). Six-week old ATH40 athymic nude mice weretreated as indicated in the legend. “Tax” denotes that paclitaxel wasinjected by i.v. as described in Table 11. D0-D30 denotes day 0 to day30 post-drug treatment. The numbers in brackets show drug concentrationsin mg/kg body weight. The body weights of ATH490 mice were normalizedbased on the total body weight on day 0 (100%). Neither CTR-17 norCTR-20 caused any notable toxic side effect to ATH490 athymic mice, asdetermined by the changes in body weights.

Neither CTR-17 nor CTR-20 was observed to cause any notable ill-effectsto mouse vital organs. The weights of four different organs (liver,spleen, kidney and lung) of ATH490 mice from different treatment groups(as described in Table 11) were measured at 30-day post-treatment.Analysis was performed using GraphPad Prism software (GraphPadSoftware). All values are presented as mean±S.E.M. Comparison betweeneach group was made by p values determined using one-way ANOVA. A pvalue of <0.05 is considered to be statistically significant. The datashows that there was no significant difference in the mass of spleen,kidney, and lung between vehicle only and drug-treated groups (p valuesfor spleen, kidney and lung were 0.99, 0.74, and 0.36, respectively).However, the liver sizes of the samples treated in combination withpaclitaxel and CTR compounds were somewhat smaller than those of thevehicle only control. (p=0.0003). Each organ weight (%) was normalizedwith total body weight (BW). This data suggests, while not wishing to belimited by theory, that the treatment of ATH490 mice with 30 mg/kg ofCTR-17 or 30 mg/kg of CTR-20 does not cause any notable ill-effects onthe animals, as determined by changes in the weights of the four vitalorgans (liver, spleen, kidney and lung) shown in FIG. 44.

FIG. 45 shows exemplary images of the effects of CTR-17 and CTR-20 onthe liver. ATH490 athymic mice were treated as indicated in the listedconcentrations for 30 days (FIG. 45A), and then the liver cellproliferation analyzed by examining the number of mitotic cells (whitearrows in FIG. 45A). “Tax” denotes paclitaxel. The numbers in thebrackets in (FIG. 45B) are mg/kg body weight. As can be seen from FIG.45 and Table 14, livers of animals treated with 10 mg paclitaxel, 30 mgCTR-17, or 30 mg CTR-20 showed small increases in the mitotic index.However, this small increase is considered normal as the AST/ALT ratiois <3 (Table 14). The small increase in mitotic cells in the livertissue was completely prevented when ½ dose of paclitaxel (5 mg) and ½dose of either CTR-17 (15 mg) or CTR-20 (15 mg) were used in combination(p<0.0001).

Neither CTR-17 nor CTR-20 was observed to cause any notable toxicity tospleen. ATH490 athymic mice were sham-treated (vehicle only) or treatedwith compounds of the indicated doses for 30 days, followed by toxicityanalysis after spleen tissues were H & E stained. The numbers in thebrackets are mg per kg of body weight. Arrows indicate the presence ofmacrophages in the red pulp (RP). Images were taken using a ZeissEPI-fluorescent microscope (10× objective). The toxicity on the spleenis summarized in Table 15. Drug administration was carried out asdescribed in Table 11. As can be seen from FIG. 46 and Table 15, unlikeanimals treated with paclitaxel (10 mg/kg), which showed considerableside effects in the spleen including an increase in cellularity,hyperplasia of myeloid and lymphoid cells, those treated with 30 mg/kgCTR-17 or 30 mg/kg CTR-20 did not show any notable ill-effects to spleentissues, except a minor increase in myeloid elements in the red pulp.Animals that were treated with a ½ dose of CTR-20 (15 mg) and a ½ doseof paclitaxel (5 mg) did not show any ill-effects to spleen.

FIG. 47 shows exemplary images of the effects of CTR-17 and CTR-20 onthe kidney. ATH490 mice were treated as described in Table 11. At day30, kidneys were harvested, stained with H&E, and observed under a ZeissEPI-fluorescent microscope (40× objective). Treatment of animals withCTR-17 (30 mg/kg), CTR-20 (30 mg/kg) or paclitaxel (5 mg/kg) combinedwith either CTR-17 (15 mg/kg) or CTR-20 (15 mg/kg) generally did notcause any notable ill-effects to the kidney. However, when mice weretreated with paclitaxel (10 mg/kg), approximately 1 out of 5 mice showedrenal abnormalities with the appearance of glassy and acellular hyalinesand hypo-cellularity with expanded space of glomeruli.

V. Discussion

With a central core composed of an aromatic ketone and an enone group,chalcone-based compounds (Scheme 3) have been reported to show potentanti-tubulin activity (Lu et al., 2012).

The binding of chalcones to tubulin was reported to be inhibited bycolchicine and podophyllotoxins, suggesting that certain chalcone-basedcompounds may effectively bind to β-tubulin through the colchicinebinding site or very close to it (Ducki et al., 2005; Ducki et al.,2009; Hadfield et al., 2003; Lawrence et al., 2000; Peyrot et al.,1992). Other studies also showed that chalcone-based compounds bind totubulin reversibly and rapidly, thus inhibiting the microtubule assembly(Stanton et al., 2011).

The development of effective and safe anticancer agents targetingmicrotubules based on a chalcone scaffold is of medicinal interest.

Ten quinolone chalcones with a range of substituents such as a nitrogroup, methoxy and methyl groups, and halogen atoms (F, Cl and Br) inthe aryl ring A were synthesized and then screened for anticanceractivity against three breast cancer cell lines and one or twonon-cancer breast cell lines. The results indicated that among thedifferent substitutions tried, 2-methoxy substitution in the aryl ring Aenhanced both the selectivity and growth inhibitory potency againstbreast cancer cells with IC₅₀ values of 0.41, 0.15 and 0.52 μM againstMDA-MB-231, MDA-MB-468, and MCF-7 cells, respectively. Hence, then 24novel quinolone chalcones were synthesized that had a 2-alkoxysubstitution in phenyl ring A (Table 1) and the anticancer activities ofsome of these compounds were then examined (Tables 2-4).

The compounds CTR-17 and CTR-20, showed preferential killing of cancerover non-cancer cells, up to 24-26 fold. Furthermore, the IC₅₀ values inkilling a variety of different cancer cell lines by CTR-17 and CTR-20were found to be in the sub-μM range, making both of them promisingleads. Further studies showed that all of the 24 novel quinolonechalcone compounds effectively kill cancer cells, many of whichpreferentially kill cancer over non-cancer cells. A study with isogeniccell lines showed that CTR-20, CTR-21 and CTR-32 preferentially kill thefully malignant MCF10CA1a breast cancer cells over premalignant MCF10AT1and non-cancer MCF10A breast cells. Previously, we identified that bothCTR-17 and CTR-20 killed two different lines of multidrug-resistantcells (overexpressing MDR1 or MRP) almost as effectively asnon-resistant cells (or better in some cases). In contrast, colchicine,paclitaxel and vinblastine are at least 10-fold less effective inkilling multidrug-resistant cells than non-resistant control cells. Itwas found that CTR-21 and CTR-32 kill the multidrug- andpaclitaxel-resistant MDA-MB231TaxR breast cancer cells with highpotency. CTR-20 also effectively kills two bortezomib-resistant multiplemyeloma cell lines (RPMI-8226-BR and ANBL6-BR), and this data indicatesthat CTR compounds effectively overcome the drug-resistant issue whichis currently a major cause of chemotherapy failure.

The data showed that both CTR-17 and CTR-20 bind to tubulin, resultingin the inhibition of microtubule polymerization. We have also shown thatthe tubulin binding sites of CTR-17 and CTR-20 closely overlap with thatof colchicine, but apart from the vinblastine binding site. Data from insilico molecular modeling suggests that CTR-17, CTR-20 and colchicine,respectively, form one, two and three H-bonds with amino acid residueson tubulin, in addition to strong Van der Waals interactions.

It is well known that agents disrupting microtubule dynamics through thebinding to the colchicine-binding site have minimal drug-resistantissues, although they tend to be quite toxic to humans. The finding thatcompounds such as CTR-20, CTR-21 and CTR-32 can overcome drug resistanceis consistent with this previous finding as at least CTR-17 and CTR-20bind to the colchicine-binding site.

In contrast to colchicine, which is known to be very toxic to humans (Luet al. 2012), compounds such as CTR-17 and CTR-20 show little toxicityto animals (FIGS. 43-47), which is consistent with the in vitro data(Table 2). Compounds such as CTR-21 and CTR-32, similarly to CTR-20,kill cells in a malignancy-dependent manner. This notion is strengthenedas the inhibition of microtubule dynamics by CTR-17, CTR-20, CTR-21 andCTR-32 is reversible upon washing off the compounds (manifested byrecovering cell cycle progression).

While not wishing to be limited by theory, the number of H-bonds betweencompounds (for example, CTR-17, CTR-20 and colchicine) and amino acidresidues of tubulin can be relevant to differences in efficacy,reversibility and toxicity. In this respect, CTR-20, which shows twoH-bonds, is quite effective on many different cancers (Tables 2 and 3)while still reversible (FIG. 17). Furthermore, CTR-20 shows preferentialcancer cell killing over non-cancer cells (Table 2). CTR-17 and CTR-20were not observed to cause DNA damage nor impede DNA replication.

Data from the experiments with mice engrafted with the MDA-MB-231metastatic breast cancer show that the efficacy of CTR-20 is almostcomparable with that of paclitaxel (although CTR-20 and paclitaxel wereused at a dose of 30 mg/kg and 10 mg/kg, respectively, the former wasgiven by i.p. and the latter was given by i.v.). However, CTR-20 is lesstoxic (FIG. 46). Further, the combination of ½ doses of CTR-20 andpaclitaxel was much more effective than a full dose of either CTR-20 orpaclitaxel alone.

Together, the in vitro data shows that novel tubulin-targetingcompounds, such as CTR-17, CTR-20, CTR-21 and CTR-32 preferentially killmany different cancer cells including all of the cell lines contained inthe NCI-60 cancer panel and the MDR1- and MRP1-overexpressingmultidrug-resistant cancer cells (which are also resistant topaclitaxel, vinblastine and colchicine). Data from mice engrafted withmetastatic breast tumor cells showed that both of these CTR compoundspossess strong antitumor activities, when used alone or in combinationwith paclitaxel.

While the present disclosure has been described with reference to whatare presently considered to be the preferred examples, it is to beunderstood that the present disclosure is not limited to the disclosedexamples. To the contrary, the present disclosure is intended to covervarious modifications and equivalent arrangements included within thespirit and scope of the appended claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety. Where a term in the present disclosure is found to bedefined differently in a document incorporated herein by reference, thedefinition provided herein is to serve as the definition for the term.

FULL CITATIONS FOR DOCUMENTS REFERRED TO IN THE SPECIFICATION

-   Borisy, G. G. and Taylor, E. W. (1967a). The mechanism of action of    colchicine. Binding of colchincine-3H to cellular protein. J. Cell    Biol. 34, 525-533.-   Borisy, G. G. and Taylor, E. W. (1967b). The mechanism of action of    colchicine. Colchicine binding to sea urchin eggs and the mitotic    apparatus. J. Cell Biol. 34, 535-548.-   Chou, T. C. (2006). Theoretical basis, experimental design, and    computerized simulation of synergism and antagonism in drug    combination studies. Pharmacol. Rev. 58, 621-681.-   Dominguez, J. N., Charris, J. E., Lobo, G., Gamboa de, D. N.,    Moreno, M. M., Riggione, F., Sanchez, E., Olson, J., and    Rosenthal, P. J. (2001). Synthesis of quinolinyl chalcones and    evaluation of their antimalarial activity. Eur. J. Med. Chem. 36,    555-560.-   Ducki, S., Mackenzie, G., Greedy, B., Armitage, S., Chabert, J. F.,    Bennett, E., Nettles, J., Snyder, J. P., and Lawrence, N. J. (2009).    Combretastatin-like chalcones as inhibitors of microtubule    polymerisation. Part 2: Structure-based discovery of alpha-aryl    chalcones. Bioorg. Med. Chem. 17, 7711-7722.-   Ducki, S., Mackenzie, G., Lawrence, N. J., and Snyder, J. P. (2005).    Quantitative structure-activity relationship (5D-QSAR) study of    combretastatin-like analogues as inhibitors of tubulin assembly. J.    Med. Chem. 48, 457-465.-   Dumontet, C. and Jordan, M. A. (2010). Microtubule-binding agents: a    dynamic field of cancer therapeutics. Nat. Rev. Drug Discov. 9,    790-803.-   Gigant, B., Wang, C., Ravelli, R. B., Roussi, F., Steinmetz, M. O.,    Curmi, P. A., Sobel, A., and Knossow, M. (2005). Structural basis    for the regulation of tubulin by vinblastine. Nature 435, 519-522.-   Hadfield, J. A., Ducki, S., Hirst, N., and McGown, A. T. (2003).    Tubulin and microtubules as targets for anticancer drugs. Prog. Cell    Cycle Res. 5, 309-325.-   Hu, C., Solomon, V. R., Ulibarri, G., and Lee, H. (2008). The    efficacy and selectivity of tumor cell killing by Akt inhibitors are    substantially increased by chloroquine. Bioorg. Med. Chem. 16,    7888-7893.-   Huang, G. S., Lopez-Barcons, L., Freeze, B. S., Smith, A. B., III,    Goldberg, G. L., Horwitz, S. B., and McDaid, H. M. (2006).    Potentiation of taxol efficacy and by discodermolide in ovarian    carcinoma xenograft-bearing mice. Clin. Cancer Res. 12, 298-304.-   Kuppens, I. E. (2006). Current state of the art of new tubulin    inhibitors in the clinic. Curr. Clin. Pharmacol. 1, 57-70.-   Lawrence, N. J., McGown, A. T., Ducki, S., and Hadfield, J. A.    (2000). The interaction of chalcones with tubulin. Anticancer Drug    Des 15, 135-141.-   Li, R., Kenyon, G. L., Cohen, F. E., Chen, X., Gong, B.,    Dominguez, J. N., Davidson, E., Kurzban, G., Miller, R. E.,    Nuzum, E. O., Rosenthal, P. J. and McKerrow, J. H. (1995). In vitro    antimalarial activity of chalcones and their derivatives. J. Med.    Chem. 38, 5031-5037.-   Lu, Y., Chen, J., Xiao, M., Li, W., and Miller, D. D. (2012). An    overview of tubulin inhibitors that interact with the colchicine    binding site. Pharm. Res. 29, 2943-2971.-   Meth-Cohn, O., Narine, B., and Tarnowski, B. (1981). A versatile new    synthesis of quinolines and related fused pyridines, Part 5. The    synthesis of 2-quinoline-3-carbaldehydes. Journal of the Chemical    Society, Perkin Transactions 1, 1520-1530.-   Peyrot, V., Leynadier, D., Sarrazin, M., Briand, C., Menendez, M.,    Laynez, J., and Andreu, J. M. (1992). Mechanism of binding of the    new antimitotic drug MDL 27048 to the colchicine site of tubulin:    equilibrium studies. Biochemistry 31, 11125-11132.-   Pryor, D. E., O'Brate, A., Bilcer, G., Diaz, J. F., Wang, Y., Wang,    Y., Kabaki, M., Jung, M. K., Andreu, J. M., Ghosh, A. K.,    Giannakakou, P., and Hamel, E. (2002). The microtubule stabilizing    agent laulimalide does not bind in the taxoid site, kills cells    resistant to paclitaxel and epothilones, and may not require its    epoxide moiety for activity. Biochemistry 41, 9109-9115.-   Ravelli, R. B., Gigant, B., Curmi, P. A., Jourdain, I., Lachkar, S.,    Sobel, A., and Knossow, M. (2004). Insight into tubulin regulation    from a complex with colchicine and a stathmin-like domain. Nature    428, 198-202.-   Singh, P., Rathinasamy, K., Mohan, R., and Panda, D. (2008).    Microtubule assembly dynamics: an attractive target for anticancer    drugs. IUBMB. Life 60, 368-375.-   Skehan, P., Storeng, R., Scudiero, D., Monks, A., McMahon, J.,    Vistica, D., Warren, J. T., Bokesch, H., Kenney, S., and Boyd, M. R.    (1990). New colorimetric cytotoxicity assay for anticancer-drug    screening. J. Natl. Cancer Inst. 82, 1107-1112.-   Srivastava, A. and Singh, R. (2005). Vilsmeier-Haack reagent: a    facile synthesis of 2-chloro-3-formylquinolines from    N-arylacetamides and transformation into different functionalities.    Indian Journal of Chemistry Section B 44, 1868.-   Stanton, R. A., Gernert, K. M., Nettles, J. H., and Aneja, R.    (2011). Drugs that target dynamic microtubules: a new molecular    perspective. Med. Res. Rev. 31, 443-481.-   Tokesi, N., Lehotzky, A., Horvath, I., Szabo, B., Olah, J., Lau, P.,    and Ovadi, J. (2010). TPPP/p25 promotes tubulin acetylation by    inhibiting histone deacetylase 6. J. Biol. Chem. 285, 17896-17906.-   Vogel, A. I., Tatchell, A. R., Furnis, B. S., Hannaford, A. J., and    Smith, P. W. G. (1996). Vogel's Textbook of Practical Organic    Chemistry. Prentice Hall).-   Weisenberg, R. C., Borisy, G. G., and Taylor, E. W. (1968). The    colchicine-binding protein of mammalian brain and its relation to    microtubules. Biochemistry 7, 4466-4479.-   Zhou, J. and Giannakakou, P. (2005). Targeting microtubules for    cancer chemotherapy. Curr. Med. Chem. Anticancer Agents 5, 65-71.

TABLE 1 Chemical names and structures. Structure Compound StructureExperiment Number (Chemical Name) Book Code 1

CTR-17 2

CTR-18 3

CTR-19 4

CTR-20 5

CTR-21 6

CTR-22 7

CTR-23 8

CTR-24 9

CTR-25 10

CTR-26 11

CTR-27 12

CTR-28 13

CTR-29 14

CTR-30 15

CTR-31 16

CTR-32 17

CTR-33 18

CTR-34 19

CTR-35 20

CTR-36 21

CTR-37 22

CTR-38 23

CTR-39 24

CTR-40

TABLE 2 Initial screening of four CTR compounds using breast cancercells (MDA-MB-231, MDA- MB-468, MCF-7) and non-cancer breast cells(184B5) determined by SRB assays. IC₅₀ (μM) ^(a,b) CODE MDA-MB-231MDA-MB-468 MCF-7 K562 HeLa 184B5^(d) MCF-10A^(d) CTR-17^(c) 0.41 ± 0.020.15 ± 0.33 0.52 ± 0.17 0.15 ± 0.10 0.33 ± 0.06 3.49 ± 0.03 3.95 ± 0.14CTR-18  0.93  0.08  0.12 0.21 ± 0.02 ND  3.89  ND^(e) CTR-19  5.11  1.91 1.93 0.95 ± 0.65 ND  3.94 ND CTR-20^(c) 0.12 ± 0.09 0.12 ± 0.02 0.14 ±0.06 0.13 ± 0.02 0.10 ± 0.02 1.24 ± 0.06 2.37 ± 0.21 Chloroquine 22.5228.58 38.44 ND ND 76.13 ND Cisplatin 23.65 31.02 25.77 ND ND 25.54 ND^(a) IC₅₀ values were calculated from Sigmoidal dose response curves(variable slope), which were generated with GraphPad Prism V. 4.02(GraphPad Software Inc.). ^(b) Values are the mean value of triplicatesof at least two independent experiments. ^(c)CTR-17 and CTR-20 wererepeated twice. ^(d)184B5 and MCF-10A are non-cancer, immortalizedbreast epithelial cell lines, and the rest are different cancer celllines. ^(e)ND, not determined.

TABLE 3 Antiproliferation effects of CTR-17 and CTR-20 on other cancercell lines. U87MG T98G NCI-H1975 A549 RPMI-8226 UC3 HEK293T (brain)(brain) (lung) (lung) (myeloma) (Bladder) (kidney) CTR-17^(b)  0.76 ±0.10^(a) 0.82 ± 0.09 0.60 ± 0.14 0.41 ± 0.06 0.36 ± 0.04 0.39 ± 0.030.42 ± 0.07 CTR-20^(b) 0.49 ± 0.13 0.22 ± 0.10 0.39 ± 0.14 0.13 ± 0.040.23 ± 0.00 0.12 ± 0.03 0.19 ± 0.00 ^(a)Numbers are IC₅₀ values in μM,determined by SRB assays as described in Table 2. ^(b)Treatment withCTR-17 and CTR-20 was for 72 hours.

TABLE 4 Antiproliferation effects of CTR compounds on cancer andnon-cancer cells as determined by SRB assays IC₅₀ ^(a, b) CODE MDA-MB231MCF7 HeLa RPMI-8226 184B5^(d) MCF10A^(d) CTR-21 (nM) 56.91 ± 10.46 75.11± 3.07  11.93 ± 1.40  5.34 ± 0.89 36.96 ± 2.5  20.32 ± 2.47  CTR-23 (μM)1.52 ± 0.12 0.50 ± 0.10 0.64 ± 0.05 1.41 ± 0.15 1.32 ± 0.03 1.12 ± 0.04CTR-24 (μM) 2.02 ± 0.01 1.54 ± 0.36 1.34 ± 0.20 1.04 ± 0.13 2.37 ± 0.192.10 ± 0.39 CTR-25 (μM) 2.60 ± 0.18 1.69 ± 0.07 1.32 ± 0.02 0.77 ± 0.212.64 ± 0.17 2.64 ± 0.15 CTR-26 (μM) 0.96 ± 0.15 0.32 ± 0.01 0.29 ± 0.020.20 ± 0.03 0.77 ± 0.11 0.91 ± 0.03 CTR-27 (μM) 2.69 ± 0.24 2.20 ± 0.351.80 ± 0.27 0.77 ± 0.10 2.80 ± 0.18 2.52 ± .03  CTR-29 (μM) 0.21 ± 0.020.10 ± 0.01 0.09 ± 0.02 0.07 ± 0.01 0.28 ± 0.01 0.27 ± 0.05 CTR-30 (μM)1.42 ± 0.08 1.47 ± 0.15 1.50 ± 0.13 0.20 ± 0.03 2.46 ± 0.14 2.79 ± 0.59CTR-32 (nM) 44.22 ± 4.21  46.36 ± 3.61  12.88 ± 0.35  6.29 ± 1.43 52.03± 7.35  30.06 ± 1.61  CTR-33 (μM) 1.36 ± 0.11 1.71 ± 0.25 0.10 ± 0.201.17 ± 0.11 4.68 ± 0.52 2.60 ± 0.73 CTR-34 (μM) 0.84 ± 0.08 0.85 ± 0.060.64 ± 0.03 0.26 ± 0.02 1.16 ± 0.10 0.98 ± 0.04 CTR-35 (μM) 2.35 ± 0.262.12 ± 0.36 2.07 ± 0.25 1.32 ± 0.10 4.29 ± 0.06 2.88 ± 0.54 CTR-36 (μM)0.73 ± 0.06 1.23 ± 0.08 1.11 ± 0.13 0.15 ± 0.02 1.84 ± 0.07 1.35 ± 0.16CTR-37 (μM) 0.39 ± 0.09 0.32 ± 0.04 0.14 ± 0.03 0.10 ± 0.03 0.44 ± 0.020.46 ± 0.11 CTR-38 (μM) 0.15 ± 0.01 0.14 ± 0.02 0.06 ± 0.01 0.04 ± 0.010.16 ± 0.02 0.11 ± 0.00 CTR-40 (μM) 0.13 ± 0.02 0.09 ± 0.01 0.04 ± 0.000.05 ± 0.02 0.10 ± 0.01 0.07 ± 0.00 ^(a) IC₅₀ values were calculatedfrom Sigmoidal dose response curves (variable slope), which weregenerated with GraphPad Prism V. 4.02 (GraphPad Software Inc.). ^(b)Values are the mean value of triplicates of at least two independentexperiments. ^(d)The 184B5 and MCF10A are non-cancer, immortalizedbreast epithelial cell lines, and the rest is cancer cell lines. *Cellswere treated for 72 hours by the CTR compounds. * All cell lines wereauthenticated on April 10, Jul. 13, 2015 & Sep. 9, 2016 (Genetica DNALaboratories) by the STR profiling of gDNA(www.celllineauthentication.com).

TABLE 5 The number of cells with a monoplar centrosome increases inresponse to CTR-17 (3 μM). 12 h post-treatment 24 h post-treatmentMonopolar^(a) ACS^(b) Monopolar ACS HeLa 80.0 ± 4.5  20.0 ± 4.5  100.0 ±0.0   0.0 ± 0.0 MDA-MB-231 40.0 ± 10.2 60.0 ± 10.2 44.9 ± 5.7 55.1 ± 5.7MDA-MB-468 55.3 ± 8.9  44.7 ± 8.9  76.0 ± 5.9 24.0 ± 5.9 HEK293T 50.5 ±10.1 48.2 ± 10.1 93.2 ± 3.4  0.1 ± 0.0 ^(a,b)The numbers in monopolarcentrosome and abnormal chromosome segregation (ACS) are percent oftotal mitotic cells.

TABLE 6 The number of cells with a monoplar centrosome increases inresponse to 1 μM CTR-20. Monopolar (%) ACA/S^(a) (%) MB231 35.73 ± 5.5958.05 ± 6.21 HeLa 48.13 ± 3.34 34.97 ± 1.96 MCF-7 62.41 ± 2.99 29.01 ±2.50 ^(a)ACA/S: Abnormal chromosome alignment and segregation.

TABLE 7 CTR-17 and CTR-20 effectively kill multidrug-resistant cells(KB-C-2 & H69AR). Numbers are IC₅₀ in nM or μM. KB-C-1 KB-C-2 Resistance(fold) SW-1271 H69AR Resistance (fold) Colchicine (nM) 5.36 ± 0.54 83.45± 7.22  15.57 4.84 ± 0.80 22.97 ± 3.63 4.74 Paclitaxel (nM) 2.01 ± 0.1723.08 ± 0.21  11.48 4.51 ± 0.71 10.99 ± 2.60 2.44 Vinblastine (nM) 0.61± 0.09 9.27 ± 3.22 15.20 1.75 ± 0.21 10.20 ± 1.97 5.82 CTR-17 (μM) 0.38± 0.07 0.65 ± 0.16 1.71 1.14 ± 0.04  0.52 ± 0.10 0.45 CTR-20 (μM) 0.10 ±0.02 0.25 ± 0.03 2.50 1.95 ± 0.01  0.13 ± 0.01 0.13 * KB-C-1 (cervicalcancer) and SW-1271 (lung cancer) cell s are multidrug naïve, and KB-C2(cervical cancer) and H69AR (lung cancer) are multidrug-resistant cancercells.

TABLE 8 The combination of CTR compounds and paclitaxel show synergisticeffects on MDR1-overexpressing KB-C-2 cells. Combination index TreatmentRatio Cell survival rates^(b) (CI) Conclusions CTR-17 only  IC₅₀ (0.65μM)  53.2 ± 12.6  NA^(a) NA 0.5IC₅₀ (0.325 μM)  80.0 ± 11.4 NA NA CTR-20only  IC₅₀ (0.25 μM) 56.8 ± 8.4 NA NA 0.5IC₅₀ (0.125 μM) 76.0 ± 9.6 NANA Paclitaxel only IC₅₀ (23 nM) 48.4 ± 5.8 NA NA 0.5IC₅₀ (11.5 nM)  75.9± 7.7 NA NA 0.25IC₅₀ (5.75 nM)  89.5 ± 6.0 NA NA 0.125IC₅₀ (2.875 nM) 99.6 ± 0.3 NA NA CTR-17 + 0.5IC₅₀:IC₅₀   15.7 ± 2.8 0.87 Moderatelysynergistic Paclitaxel 0.5IC₅₀:0.5IC₅₀ 29.2 ± 7.5 0.79 Moderatelysynergistic IC₅₀:IC₅₀  4.7 ± 1.6 0.71 Substantially synergistic  IC₅₀:0.5IC₅₀ 12.1 ± 3.6 0.76 Moderately synergistic    IC₅₀:0.25IC₅₀18.3 ± 6.2 0.73 Substantially synergistic    IC₅₀:0.125IC₅₀ 26.9 ± 9.20.77 Moderately synergistic CTR-20 + 0.5IC₅₀:IC₅₀   16.3 ± 2.6 0.72Substantially synergistic Paclitaxel 0.5IC₅₀:0.5IC₅₀ 39.1 ± 6.1 0.89Moderately synergistic IC₅₀:IC₅₀  9.3 ± 1.9 0.69 Substantiallysynergistic   IC₅₀:0.5IC₅₀ 16.0 ± 2.0 0.69 Substantially synergistic   IC₅₀:0.25IC₅₀ 31.4 ± 2.9 0.86 Moderately synergistic   IC₅₀:0.125IC₅₀ 42.7 ± 3.0 0.95 Slightly synergistic ^(a)NA, notapplicable ^(b)Values are the mean value of triplicates of at least fourindependent experiments.

TABLE 9 The combination of CTR compounds and paclitaxel show synergisticeffects on MDR1-overexpressing MDA-MB231TaxR cells. Treatment Ratio Cellsurvival rate^(a) CI^(b) Conclusion CTR-20 only  0.3 μM  63.7 ± 2.52 NA^(c) NA CTR-21 only 23.4 nM 52.10 ± 3.71 NA NA CTR-32 only 23.4 nM53.07 ± 6.50 NA NA Paclitaxel only  300 nM 57.75 ± 3.78 NA NA  150 nM82.41 ± 5.00 NA NA 75.0 nM 90.98 ± 4.49 NA NA 37.5 nM 90.28 ± 3.58 NA NA18.75 nM  95.84 ± 1.86 NA NA CTR-20 + 0.3 μM + 300 nM  33.27 ± 1.86 0.96Additive paclitaxel 0.3 μM + 150 nM  37.97 ± 3.02 0.72 Moderatelysynergistic 0.3 μM + 75.0 nM 40.66 ± 3.88 0.63 Synergistic 0.3 μM + 37.5nM 44.37 ± 1.38 0.58 Synergistic CTR-21 + 23.4 nM + 300 nM  32.31 ± 2.490.55 Synergistic paclitaxel 23.4 nM + 150 nM  39.05 ± 2.04 0.53Synergistic 23.4 nM + 75.0 nM  43.30 ± 4.93 0.56 Synergistic 23.4 nM +37.5 nM  41.80 ± 1.92 0.38 Synergistic 23.4 nM + 18.75 nM 42.24 ± 0.930.35 Synergistic CTR-32 + 23.4 nM + 300 nM  35.90 ± 0.55 0.61Synergistic paclitaxel 23.4 nM + 150 nM  36.92 ± 1.13 0.36 Synergistic23.4 nM + 75.0 nM  44.03 ± 1.51 0.35 Synergistic 23.4 nM + 37.5 nM 46.18 ± 0.42 0.30 Synergistic 23.4 nM + 18.75 nM 47.76 ± 0.28 0.27Synergistic ^(a)Values are the mean value of triplicates of at leastthree independent experiments ^(b)CI: Combinational index. ^(c)NA: notapplicable

TABLE 10 The combination of CTR-20 and ABT-737 shows strong synergisticeffects against MDA-MB231 cells. Treatment Ratio Cell survival rate^(a)CI^(b) Conclusion ABT-737 only 6.25 μM  91.01 ± 9.81  NA^(c) NA 3.125μM  98.61 ± 3.93 NA NA CTR-20 only 0.4 μM 26.38 ± 4.73 NA NA 0.2 μM41.07 ± 4.58 NA NA 0.1 μM 70.86 ± 4.17 NA NA CTR-20 + ABT-737 0.4 μM +6.25 μM  −15.16 ± 2.11  0.10 Strongly synergistic 0.2 μM + 6.25 μM −10.41 ± 2.84  0.07 Very strongly synergistic 0.1 μM + 6.25 μM  42.30 ±3.38 0.57 Synergistic 0.4 μM + 3.125 μM −10.98 ± 3.17  0.09 Verystrongly synergistic 0.2 μM + 3.125 μM 13.18 ± 4.91 0.26 Stronglysynergistic 0.1 μM + 3.125 μM 61.82 ± 3.98 0.84 Moderately synergistic^(a)Values are the mean value of triplicates of at least threeindependent experiments ^(b)CI: Combinational index. ^(c)NA: notapplicable

TABLE 11 Typical protocol for the study of CTR compounds using xenograftmice (ATH490). Treatment Dosage Frequency Route Notes Sham controlHighest volume Every 3 days Intraperitoneal (I.P.) Vehicle onlyPaclitaxel 10 mg/kg B.W.^(a) Once/week Intravenous (I.V.) CTR-17 (30) 30mg/kg B.W. Every 3 days I.P. CTR-20 (30) 30 mg/kg B.W. Every 3 days I.P.Paclitaxel (5), Paclitaxel 5 mg/kg B.W. & Once/week Paclitaxel (I.V.) &Paclitaxel was given 24 CTR-17 (15) CTR-17 15 mg/kg B.W. CTR-17 (I.P.)hours prior to CTR-17 Paclitaxel (5), Paclitaxel 5 mg/kg B.W. &Once/week Paclitaxel (I.V.) & Paclitaxel was given 24 CTR-20 (15) CTR-2015 mg/kg B.W CTR-20 (I.P.) hours prior to CTR-20 ^(a)B.W. denotes bodyweight.

TABLE 12 Antitumor activity of CTR-17 and CTR-20, alone or incombination with paclitaxel. Day 0 Day 6 Day 14 Day 17 Day 20 Day 24 Day27 Day 30 Sham control 89.61 ± 105.03 ± 136.66 ± 162.62 ± 192.69 ±268.08 ± 426.87 ± 557.66 ± 8.97 15.90 16.87 15.29 16.56 37.85 7.57 24.72Tax^(a) (10 mg^(b)) 91.84 ± 90.11 ± 94.66 ± 92.33 ± 81.66 ± 95.61 ±139.41 ± 170.36 ± 6.52 17.42 31.77 31.56 30.09 29.69 24.96 40.07 CTR-17(30 mg) 86.71 ± 63.99 ± 90.07 ± 128.36 ± 134.76 ± 160.86 ± 189.45 ±209.84 ± 5.42 3.85 21.30 20.65 26.46 37.30 47.61 56.45 CTR-20 (30 mg)92.51 ± 69.89 ± 84.64 ± 97.11 ± 97.22 ± 106.26 ± 124.88 ± 140.63 ± 10.458.22 5.09 8.59 13.40 26.05 34.85 38.00 Tax (5 mg) plus 91.38 ± 74.70 ±70.05 ± 65.42 ± 60.67 ± 70.80 ± 81.88 ± 108.37 ± CTR-17 (15 mg) 13.0017.35 13.74 23.79 21.29 17.46 19.85 35.30 Tax (5 mg) plus 95.35 ± 61.76± 65.65 ± 51.14 ± 54.36 ± 51.19 ± 47.20 ± 65.71 ± CTR-20 (15 mg) 3.465.76 16.32 12.37 8.13 6.90 13.15 22.00 ^(a)Tax: paclitaxel. ^(b)mg perkg of body weight.

TABLE 13 Antitumor activity of CTR-18 and CTR-19. D 0 D 6 D 14 D 17 D 20D 24 D 27 D 30 Control 89.61 ± 105.03 ± 136.66 ± 162.62 ± 192.69 ±268.08 ± 426.87 ± 557.66 ± 8.97 15.90 16.87 15.29 16.56 37.85 7.57 24.72CTR-18 (30 mg) 92.51 ± 111.09 ± 138.10 ± 145.48 ± 166.84 ± 143.78 ±141.52 ± 253.37 ± 6.73 23.55 35.18 34.28 48.43 23.27 16.66 77.10 CTR-19(30 mg) 92.51 ± 84.79 ± 99.68 ± 92.27 ± 115.39 ± 124.76 ± 143.62 ±200.77 ± 12.97 10.38 9.41 8.19 8.62 9.48 20.22 50.95 Tax^(a) (5 mg^(b))plus 84.60 ± 78.81 ± 96.61 ± 88.40 ± 92.31 ± 109.97 ± 125.23 ± 178.95 ±CTR-18 (15 mg) 7.36 4.61 12.22 4.90 16.20 20.81 19.03 29.63 Tax (5 mg)plus 93.84 ± 96.25 ± 102.59 ± 97.72 ± 101.44 ± 143.28 ± 189.18 ± 206.37± CTR-19 (15 mg) 8.15 5.15 13.81 7.98 18.94 28.02 41.47 44.05 ^(a)Tax:paclitaxel. ^(b)mg per kg of body weight.

TABLE 14 Analysis of liver toxicity by AST and ALT. Treatment ALT^(a)(IU/L) AST^(b)(IU/L) Untreated 54.08 ± 4.98 113.08 ± 9.13  Sham Control64.03 ± 4.04 112.46 ± 4.74  Paclitaxel (10 mg/kg) 52.02 ± 2.59 111.14 ±13.42 CTR-17, 20 mg/kg 63.59 ± 9.73 101.00 ± 10.19 CTR-17, 30 mg/kg64.67 ± 5.26 114.13 ± 6.36  CTR-20, 20 mg/kg 56.77 ± 5.34 119.19 ± 4.03 CTR-20, 30 mg/kg 50.50 ± 9.96 118.22 ± 17.00 Paclitaxel (5), CTR-17 (15)46.01 ± 0.42 106.70 ± 13.41 Paclitaxel (5), CTR-20 (15) 59.62 ± 7.25107.67 ± 13.22 ^(a)ALT: Alanine transaminase. ^(b)AST: aspartateaminotransferase.

TABLE 15 Toxicology analysis of spleen. Treatment White pulp (WP) RedPulp (RP) Capsule Untreated Normal Normal Normal Vehicle Control NormalNormal Normal Paclitaxel (10 mg/kg) Less but bigger Myeloid Normalelements: ↑↑^(a) CTR-17, 30 mg/kg Less but bigger Myeloid Normalelements: ↑ CTR-20, 30 mg/kg Less but bigger Myeloid Normal elements: ↑Tax (5), CTR-17 (15) Normal Normal Normal Tax (5), CTR-20 (15) NormalNormal Normal ^(a)Double and single upward arrows indicate highly andmoderately increased, respectively

What is claimed is:
 1. A compound of Formula I:

wherein A is O or S; n is 0, 1, 2 or 3; when n is 1, R¹ is halo,C₁₋₆alkyl, C₂₋₆alkenyl or —X—C₁₋₆alkyl; when n is 2 or 3, each R¹ isindependently halo, C₁₋₆alkyl, C₂₋₆alkenyl or —X—C₁₋₆alkyl; or two R¹together form a methylenedioxy group that is attached to two adjacentring carbon atoms; R² is C₁₋₆alkyl or C₁₋₆haloalkyl; R³ is absent or ishalo, —X—C₁₋₆alkyl or —X—C₁₋₆haloalkyl; and each X is independently O orS, or a pharmaceutically acceptable salt, solvate and/or prodrugthereof.
 2. The compound of claim 1, wherein A is O.
 3. The compound ofclaim 1, wherein R³ is absent.
 4. The compound of claim 1, wherein R² ismethyl.
 5. The compound of claim 1, wherein n is
 0. 6. The compound ofclaim 1, wherein n is 1 and R¹ is 6-OCH₃, 7-OCH₃, 8-OCH₃, 6-OC₂H₅,6-SCH₃, 7-SCH₃, 6-CH₃, 6-C₂H₅, 6-F, 6-Cl, 6-Br, 7-F, 7-Cl or 7-Br,optionally wherein R¹ is 6-CH₃, 6-OCH₃ or 7-OCH₃.
 7. The compound ofclaim 1, wherein n is 2 and R¹ is 6,7-diCH₃, 6,7-diOCH₃ or 6,7-O—CH₂—O—.8. The compound of claim 1, wherein n is 3 and R¹ is 5,6,7-triOCH₃. 9.The compound of claim 1, wherein the compound is selected from:

or a pharmaceutically acceptable salt, solvate and/or prodrug thereof.10. The compound of claim 9, wherein the compound is:


11. The compound of claim 9, wherein the compound is:


12. A pharmaceutical composition comprising one or more compounds ofclaim 1 and a pharmaceutically acceptable carrier.
 13. A method oftreating cancer comprising administering one or more compounds of claim1 to a subject in need thereof, wherein the cancer is breast cancer,leukemia, cervical cancer, brain cancer, lung cancer, bladder cancer,kidney cancer, multiple myeloma or other blood cancers, colorectalcancer, CNS cancer, melanoma, ovarian cancer or prostate cancer. 14.(canceled)
 15. The method of claim 13, wherein the cancer comprisescolchicine-resistant, paclitaxel-resistant, bortezomib-resistant,vinblastine-resistant and/or multidrug-resistant tumor cells.
 16. Themethod of claim 13, wherein the one or more compounds of claim 1 areadministered in combination with one or more other anticancer agents.17. The method of claim 16, wherein the other anticancer agents areselected from the group consisting of mitotic inhibitors, optionallypaclitaxel; bcl2 inhibitors, optionally ABT-737; proteasome inhibitors,optionally bortezomib or calfilzomib; signal transduction inhibitors,optionally gefitinib, erlotinib, dasatinib, imatinib or sunitinib;inhibitors of DNA repair, optionally iniparib, temozolomide ordoxorubicin; and alkylating agents, optionally cyclophosphamide.
 18. Themethod of claim 17, wherein the other anticancer agent is paclitaxel.19. The method of claim 16, wherein the dosage of the one or morecompounds of claim 1 is less than the dosage of the one or morecompounds of claim 1 when administered alone.
 20. The method of claim19, wherein the dosage of the one or more compounds of claim 1 is onehalf the dosage of the one or more compounds of claim 1 whenadministered alone.
 21. The method of claim 16, wherein the dosage ofthe other anticancer agent is less than the dosage of the otheranticancer agent when administered alone.
 22. The method of claim 21,wherein the dosage of the other anticancer agent is one half the dosageof the other anticancer agent when administered alone.